Vectors based on recombinant defective viral genomes and their use in the formulation of vaccines

ABSTRACT

The vectors comprise a recombinant defective viral genome expressing at least one antigen suitable for the induction of systemic and secretory immune responses or an antibody conferring protection against an infectious agent. The defective viral genome comprises the genome of a parental virus having the viral replicase recognition signals located on ends 3′ and 5′, further comprising internal deletions, and wherein said defective viral genome depends on a helper virus for its replication and encapsidation. These vectors are suitable for the forming of a recombinant system comprising the aforesaid expression vector, and a helper virus. The system is suitable for the manufacture of mono- and polyvalent vaccines against infectious agents of different animal species, especially pigs, dogs and cats, and as expression vehicles for antibodies protective against infectious agents.

[0001] The vectors comprise a recombinant defective viral genome expressing at least one antigen suitable for the induction of systemic and secretory immune responses. The defective viral genome comprises the parental virus genome having viral replicase recognition signals located on ends 3′ and 5′ further comprising internal deletions, and wherein said defective viral genome depends on a helper virus for its replication. These vectors are suitable for the forming of a recombinant system comprising (a) the aforesaid expression vector, and (b) a helper virus which supplies functional and structural proteins for the replication and encapsidation of the defective genome. This system is suitable for the manufacture of mono- and polyvalent vaccines against infectious agents of various animal species, especially pigs, dogs and cats.

FIELD OF THE INVENTION

[0002] This invention relates to a number of vectors based on recombinant defective virus genomes expressing antigens suitable for the induction of systemic and secretory immune responses for the prevention of infections in the mucosae, and to their use with vaccinal purposes together with a suitable helper virus.

BACKGROUND OF THE INVENTION

[0003] The attainment of recombinant proteins using expression vectors is a well-known fact. In general, prokaryotic and yeast expression systems are highly efficacious and easy to use, whereas the expression systems used containing superior eukaryotic cells present a number of drawbacks relative to low protein production levels and limitations in the host range. Of the existing expression systems for superior eukaryotic cells, baculovirus-based vectors are the most efficacious in terms of protein production. However, they can only be used in insect cells that, as is known, glycosilate proteins differently from the way animal cells do. In addition, the construction of the recombinant virus takes place through a homologous recombination, which is a laborious technique, especially when numerous genetic variants have to be analyzed.

[0004] On the other hand, vectors based on DNA viruses suitable for heterologous gene expression are known. However, the use of DNA-based vectors presents numerous drawbacks for, as they replicate in the nucleus of the host cell and can become integrated in the genome, they are therefore not reliable. On the contrary, the use of RNA-based vectors overcomes the drawbacks associated with the use of DNA viruses because, since they replicate not in the genome of the host cell but in the cytoplasm, replication takes place via RNA and not via DNA, and the possibilities of integration in the genome are very low, making the vectors based on these RNA viruses more reliable.

[0005] Also well-known are defective interfering particles (DI) containing the virion capsid and a defective genome, which are deletion subgenomic mutants mostly generated form infectious viral genomes by a replication error. In general, the term “DI particle” refers to defective viruses lacking a region of the RNA or DNA genome, containing the proteins and antigens of the virus, requiring co-infection of the infectious parental virus (helper virus) for replication and which specifically interfere with the homologous helper virus, as they replicate at its expense [Huang and Baltimore, Nature, 226, 325-327 (1970)]. DI genomes arise from genome reorganizations as a result of shifts of the RNA polymerase from one RNA template to another or from one segment of an RNA template to another segment of the same molecule. These DI genomes, once they have been generated, self amplify at the expense of the parental genome or the amplifying virus coding for the proteins involved in replication and encapsidation and which has to compete with defective genomes for such products.

[0006] DI particles have been obtained and characterized from some coronaviruses, such as the murine hepatitis virus (MHV), infectious bronchitis virus (IBV), and bovine coronavirus (BCV), although DI particles derived from porcine transmissible gastroenteritis virus (TGEV) have not been described. One of the MHV natural DI particles has been used as the basis for the development of an expression vector in which the exogenous gene is inserted under the control of an internal promoter transcription sequence [Lin and Lai, J. Virol., 6110-6118, October (1993)].

[0007] Generally, known heterologous gene expression vectors based on DI particles have some drawbacks related with their species and target organ specificity and their limited capacity for cloning, that limit their possibilities of use, both in basic research and in research applied to the development of such vectors for therapeutical purposes, including vaccines.

[0008] Consequently, there is still need of heterologous gene expression vectors that may successfully overcome the mentioned drawbacks. Specifically, it would be highly advantageous to have available some heterologous gene expression vectors with a high level of safety and cloning capacity and which could be designed so that their species specificity and tropism might be easily controlled.

[0009] The present invention provides a solution to the existing problem, comprising a vector based on a recombinant defective viral genome expressing antigens suitable for the induction of an immune response and for the prevention of infections caused by different infectious agents in various animal species. The heterologous gene expression vectors (or DNA sequences) provided by this invention have a high level of safety, as well as high cloning capacity, and may be designed so that their species specificity and tropism may easily be controlled, making such vectors suitable for the formulation of vaccines capable of conferring protection against infections caused by the different infectious agents in various animal species.

[0010] Therefore, an object of the present invention is a vector based on a recombinant defective viral genome expressing at least one antigen suitable for the induction of immune response—specifically, a systemic and secretory immune response against infectious agents in various animal species—, or an antibody providing protection against an infectious agent provided with a high level of safety and cloning capacity, and which may be designed so that its species specificity and tropism may be easily controlled.

[0011] The defective viral genome which serves as a basis for the construction of the said vector is also an additional objective of this invention.

[0012] Another additional object of this invention is a recombinant expression system of heterologous proteins comprising (a) the vector described above and (b) a helper virus that will provide the proteins involved in the replication and encapsidation of the recombinant defective viral genome.

[0013] Another additional objective of this invention is vaccines capable of inducing protection against infections caused by different infectious agents in various animal species, comprising the recombinant system described above, together with a pharmaceutically acceptable excipient. These vectors can be uni-, or multivalent, depending on whether the expression vectors present in the recombinant system express one, or more antigens capable of inducing an immune response against one, or more infectious agents, or one or more antibodies providing protection against one or more infectious agents.

[0014] Other objects provided by this invention comprise a method for the immunization of animals, consisting of the administration of the said recombinant system or vaccine, as well as a method for the protection of newborn animals from infectious agents that infect the mentioned species.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1 shows the structure of TGEV. The virion is a spherical particle consisting of a lipidic envelope in whose interior is a single-chain, positive-polarity RNA molecule of 28.5 kilobases (kb). This RNA is associated to an N protein forming the nucleocapsid. M and sM structural proteins are included in the membrane. Protein S groups itself into trimers, and is anchored on the external part of the envelope forming the peplomers.

[0016]FIG. 2 shows the genomic organization of the four sequenced coronaviruses: MHV, IBV, HCV229E (human coronavirus 229E) and TGEV. The open reading frames coding for each protein are shown to scale. In each genome, the beginning of the RNAs expressing each virus is indicated with an arrow. The number of messenger RNAs (mRNA) expressed by viruses MHV or TGEV may vary depending on the viral strain. In this outline, the TGEV arrows correspond to the mRNAs expressed by strain THER-1. The mRNAs are 3′-coterminal and are numbered in decreasing size order.

[0017]FIG. 3 shows the expression of the TGEV genome, strain THER-1. The disposition of the open reading frames (ORF) in the genome is indicated: Pol, polymerase; S, sM, M and N, structural proteins; nsp 3a, 3b and 7, non-structural proteins (protein 3b is not produced with this virus). The genome is transcribed in an RNA of equal length but of negative polarity (−) that will serve as a template for the synthesis of the 7 mRNAs (1 to 7). In each mRNA are represented the common sequence, leader, of the 5′ end (square), the polyadenine section at the 3′ end and the zone which is translated in each one of them (thick lines).

[0018]FIG. 4 shows the evolution of the titer of TGEV THER-1 (A) and PUR46-mar 1CC12 (B) isolates with the passage number at high multiplicity of infection (m.o.i.) in ST cells.

[0019]FIG. 5 shows the results of the electrophoretic analysis of the RNAs produced in ST cells infected with THER-1 virus passaged 46 times at high m.o.i. The passage number is indicated above each lane and the bars to the left indicate the position of the us molecular weight markers (genomic RNA of the TGEV and GibcoBRL markers), expressed in kb. The bars to the right indicate the TGEV mRNAs and the defective interfering (DI) RNAs. NI, not infected.

[0020]FIG. 6 shows the results of the Northern-blot assay of the RNA of ST cells infected with the THER-1p35 virus.

[0021]FIG. 7 shows the results of the Northern-blot assay of the RNA proceeding from diluted passages of the THER-1-STp41 virus in ST cells.

[0022]FIG. 8 shows the effect of the cell line change on the propagation of defective RNAs A, B and C. Virus THER-1-STp46 was passaged ten times undiluted in IPEC (intestinal pig epithelium cells), and five times in PM (porcine macrophage) cells. FIGS. 8A and 8B: evolution of the virus titer with the number of the passage in IPEC and PM, respectively. FIG. 8C: analysis of the RNA of ST cells infected with virus from passages 1 and 10 in IPEC (by metabolic labeling with 32P_(i)), or of passages 1 and 5 in PM cells (by hybridization with one oligonucleotide complementary to the leading RNA).

[0023]FIG. 9 shows the encapsidation of defective genomes A, B and C. FIG. 9A shows an agarose gel stained with ethidium bromide, in which the RNA extracted from purified virions of passage 1 and passage 41 are analyzed by means of centrifugation through a 15% sucrose cushion (w/v). In the lane of passage 41, RNAs A, B and C can be observed, as well as the parental genome. The bars on the left indicate mobility markers in kb. FIG. 9B shows the results of the analysis of the RNA of passage 41 virions purified by centrifugation through sucrose cushions or continuous gradient, by Northern-blot assay with an oligonucleotide complementary to the leader RNA. Commercial GibcoBRL RNAs and RNA from passage 1 virions (lane a) were used as markers. Lanes b and c, RNA extracted from virions sedimented through 31% and 15% sucrose cushions (w/v), respectively. Lanes d and e, RNA extracted from virus purified through a continuous sucrose cushion, fractions of 1.20 and 1.15 g/ml density, respectively.

[0024]FIG. 10 shows the strategy for the cloning of defective RNAs DI-B and DI-C, in which a schematic representation can be observed of the complementary DNA fragments (cDNA) obtained by RT-PCR, using as template genomic RNA of total length (A), DI-B (B) and DI-C (C). The dotted lines indicate absence of the anticipated fragment due to its large size. The defective RNAs were cloned into four overlapping fragments (a, b, c and d), represented by lines; the numbers below these lines indicate fragment size determined in agarose gels. The oligonucleotides used as primers and their polarity are indicated by means of arrows and numbers. Oligonucleotide sequence is shown in Table 2. Striped or open boxes in (A) indicate the relative position of the viral genes: pol, polymerase; S, M and N, structural genes; 3a, 3b, sM and 7, small ORFs. The shaded thin boxes indicate the leader sequence.

[0025]FIG. 11 shows the results of the electrophoretic analysis of PCR products obtained in the amplification of defective RNAs. The RNA of purified virions THER-1p1 or THER-1p41 was used as a template in an RT-PCR reaction with oligonucleotides 1 and 2 (a), 3 and 4 (b), 5 and 6 (c), or 7 and 8 (d), whose sequence and position in the parental genome is indicated in Table 2. The lane corresponding to the RNA template of passage 1 (parental genomic RNA) or of passage 41 (parental RNA, DI-A, DI-B and DI-C), and the lane of DNA mobility markers (M, GibcoBRL) are indicated in each case. The numbers in bold type indicate the size in kb of the amplification products specific for defective RNAs. RNAs B+C, RNAs B and C: purified bands in an experiment where the RNAs of THER-1 STp41 virus were fractionated in gel. RNAs B and C migrate very close, and were cut as a single band.

[0026]FIG. 12 shows the complete RNA DI-C cDNA sequence (see SEC. DI No. 24) obtained by the sequencing of overlapping fragments of the a, b, c and d cloning. RNA DI-C has kept four discontinuous parental genome regions: I, II, III and IV. The flanking sites of these regions are indicated with arrows. The translation of the three ORFs present in genome DI-C is indicated: chimeric ORF of 6.7 kb resulting from the fusion of discontinuous regions I and II in phase; the mini-ORF of three amino acids preceding it in phase, and the ORF which initiates in the AUG of gene S. Highly homologous regions—with the proteic domains described for other coronaviruses as those responsible for the polymerase and helicase activities, and metal ion binding sites—appear shaded. CTAAAC transcription promoter sequences appear shaded. The overlapping area between ORFs 1a and 1b (41 nucleotides) appears shaded, the slippery sequence of the ribosome appears underlined, and the ORF1a termination codon in a box. In positions 637, 6397 and 6485 the specific changes with respect to the parental genome are indicated. The nucleotides present in the parental genome in these positions are indicated.

[0027]FIG. 13 shows a diagram of the RNA DI-C structure. Total genomic length appears to the right of the boxes. RNA DI-C contains four discontinuous TGEV genome regions (I, II, III and IV). These regions comprise 2.1 kb of the 5′ end of the genome, almost complete ORF1b including the overlapping area between ORFs 1a and 1b, the initiation of gene S, incomplete ORF7 and untranslated region 3′. The letters and numbers above the parental genome box indicate viral genes. The numbers below the box indicate the position of the flanking nucleotides of the discontinuous regions in the parental genome, taking as reference the sequence of the TGEV PUR46-PAR isolate. In the box corresponding to RNA DI-C, the length of the four discontinuous regions is indicated in nucleotides. In the third box it is indicated the number of nucleotides derived from each viral gene, taking into account that ORFs 1a and 1b overlap with each other 43 nucleotides in the parental genome. The QRFs predicted in the computer analysis are indicated with arrows or arrowheads. Pnt, pseudoknot; Pol, polymerase; Mib, metal ion binding; Hel, helicase; Cd, conserved domain.

[0028]FIG. 14 shows the structure of the RNA DI-B. RNA DI-B contains three discontinuous regions (I, II and III) of the TGEV genome, comprising 2.1 kb of the 5′ end of the genome, complete ORF1b including the overlapping area between ORFs 1a and 1b, the initiation of gene S, the termination of ORF7, and the untranslated region of 3′ end. Letters and numbers over the parental genome box indicate viral genes. The numbers underneath the box indicate the position in the parental genome of the flanking nucleotides of the discontinuous regions, taking as reference the sequence of the TGEV PUR46-PAR isolate. Size heterogeneity of the deletion occurring between discontinuous regions II and III is responsible for the actual existence of a DI-B genome population. In the second and third boxes are indicated the length in nucleotides of the three discontinuous regions for the largest and smallest sized genomes, respectively. In the third box it is indicated the number of nucleotides derived from each viral gene, taking into account that ORFs 1a and 1b overlap each other for 43 nucleotides in the parental genome. The ORFs predicted by the computer analysis are indicated with arrows or arrowheads. Pnt, pseudoknot; Pol, polymerase; Mib, metal ion binding; Hel, helicase; Cd, conserved domain.

[0029]FIG. 15 shows the secondary and tertiary RNA structures in the overlapping zone between ORFs 1a and 1b in the RNA DI-C. (A) Structure predicted when considering the region closest to the fork-like structure presenting complementarity to the nucleotides of the knot thus constituting the pseudoknot (nucleotides 2354 to 2358). The slippery sequence UUUAAAC is underlined. ORF1a termination codon is indicated in the box. (B) Schematic representation of this pseudoknot, which involves two sequence complementarity sections (stems: S1 and S2). The slippery sequence is reperesented in a box. (C) An alternative model taking into account the sequence of nucleotide 2489 to 2493 in the folding of the pseudoknot. This structure contains an additional complementarity sequence (stem) section. (D) Schematic representation of the pseudoknot, in which the three stems are marked: S1, S2 and S3.

[0030]FIG. 16 shows the mapping of RNAs DI by hybridization with oligonucleotides specific to the virus in Northern-blot assays. The RNA of THER-1-STp41 virus was fractionated in agarose gels until a clear separation of the parental genome RNAs and DI A, B and C was obtained. The RNA was transferred to nylon filters which were hybridized with several oligonucleotides labeled with ³²P_(i), hybridized with the parental genome (+), and hybridized (+) or not (−) with the defective genomes. The approximate localization of the sequences complementary to the oligonucleotides in the parental genome is indicated by arrows. Their exact sequence and position are indicated in Table 3. All the oligonucleotides hybridized with the parental genome and gave the expected results with RNAs B and C.

[0031]FIG. 17 shows an outline of the method for the obtainment of vaccinal viruses by transfection of infected cells with RNA DI-C. A prototype outline is illustrated with the construction that enabled the production of DI-C RNA by in vitro transcription, maintaining the 5′ and 3′ ends present in the original defective particle. The sequence of the T7 promoter [PrT7] and the sequence of the autocatalytic ribozyme of the hepatitis delta virus (HDV) [Rz HDV] were cloned flanking the DI-C RNA sequence. The position of the autocatalytic cleavage introduced by the ribozyme is marked above the sequence. The arrows indicate the positions of the internal transcription promoter sequences maintained in a natural form in the RNA DI-C. L, leader. T7ø, T7 bacteriophage transcription termination signals. Virions encapsidating both the helper virus as well as the defective genomes in which the heterologous genes had been cloned were recovered, when the in vitro transcribed RNAs were transfected into ST cells infected with the corresponding helper virus.

[0032]FIG. 18 shows a prototype outline of the construction that enabled the production of pDIA-6A.C3 by in vitro transcription, maintaining the 3′ and 5′ ends present in the original defective particle. The sequence of the bacteriophage T7 promoter [PrT7] and the presence of the autocatalytic ribozyme of the hepatitis delta virus (HDV) [RzHDV] were cloned flanking the cDNA sequence coding for an autoreplicative RNA. Plasmid pDIA-6A.C3 contains the gene coding for monoclonal antibody 6A.C3 which neutralizes TGEV [see Example 4]. The cloning of the heterologous gene was done after ORF1b, following the S gene initiation codon (AUG), and in reading phase with this gene (IGS: intergenic sequence; L: leader sequence; D: diversity region; J: joining region; VH: variable module of the immunoglobulin heavy chain; CH: constant module of the immunoglobulin heavy chain; VK: variable module of the immunoglobulin light chain; CK: constant module of the immunoglobulin light chain; polyA: polyA sequence; T7ø: T7 transcription terminator].

DETAILED DESCRIPTION OF THE INVENTION

[0033] This invention provides heterologous DNA expression vectors, based on recombinant defective viral genomes expressing, at least, an antigen suitable for the induction of immune response against various infectious agents of different animal species, or an antibody affording protection against an infectious agent, provided with high safety and cloning capacity, and which may be designed so that their species specificity and tropism may be easily controlled.

[0034] The term “infectious agent” in the sense in which it is used in this description includes any viral, bacterial or parasitic infectious agent that can infect an animal and cause a pathology.

[0035] The term “animal species” includes animals of any species, usually mammalians, and swine, canine or feline in particular.

[0036] In a specific realization of this invention, expression vectors are obtained based on recombinant defective viral genomes expressing antigens suitable for the induction of systemic and secretory immune responses, for the prevention of mucosal infections, designed to enable an easy control of species specificity and their tropism for the infection of enteric or respiratory mucosae, which makes them quite adequate for the induction of mucosal and lactogenic immunity, of particular interest in the protection of newborn animals against infections of the intestinal tract. In another specific realization of this invention, an expression vector is provided based on a recombinant defective viral genome expressing, at least, one antibody which affords protection against an infectious agent.

[0037] The expression vectors obtained by means of this invention comprise a defective viral genome derived from a virus, preferably, a virus with RNA genome and positive polarity, that maintains the 3′ and 5′ ends of the parental virus, has internal deletions and depends on a helper virus for its replication. Therefore, the invention provides, additionally, a defective virus genome comprising the genome of a parental virus having viral replicase recognition signals located at the 3′ and 5′ ends, comprising also internal deletions, and in which the aforesaid defective viral genome depends on a helper virus supplying the functional and structural proteins for the replication and encapsidation of the recombinant defective viral genome. In a specific realization, the defective viral genome comprises, additionally, the complete sequence coding for the parental virus replicase. In this case, if so desired, the helper virus can provide only the structural proteins required for the encapsidation of the recombinant defective viral genome or, alternatively, the functional and structural proteins for the replication and encapsidation of the recombinant defective viral genome. When the virus from which the recombinant defective genome derives is a virus with RNA genome, the expression vector comprises the cDNA complementing the aforesaid defective RNA or a cDNA substantially complementing the aforesaid defective RNA.

[0038] The vectors provided by this invention have a high cloning capacity—at least 18 kb—which is the greatest cloning capacity described for a vector based on RNA eukaryotic viruses. Additionally, these vectors have a high safety level because they (a) are based on defective genomes, (b) comprise RNA genomes and do not use DNA as a replicative internediary, and c) are based on viruses growing in the cytoplasm of infected cells, all of which prevents the defective genome from recombining with the cell chromosome.

[0039] In a specific realization of this invention, the obtainment of defective RNA genomes derived from coronaviruses, in particular from TGEV, is described. These genomes have the additional advantage of a very low TGEV recombination frequency (<1×10⁹), which prevents the defective genome from recombining easily with the helper virus genome. However, even though this recombination might actually take place, an attenuated virus would be obtained since the invention contemplates the convenience of using the same attenuated virus both as helper virus as well as starting material for the obtainment of the defective genome.

[0040] The defective genomes constituting the basis of such vectors can be obtained in different cell systems by means of undiluted serial passages of the virus from which they derive. The DI particle generation frequency can vary much in different virus-cell systems; for this reason it is advisable to make the passages with different virus isolates in different cell lines with the aim of selecting the proper isolate and cell line. After a certain number of passages, the viruses are isolated and then used to analyze the intracellular RNAs produced in the infection with the purpose of observing the possible appearance of bands not corresponding to any viral mRNA, in which case, in order to analyze the nature of these new RNAs—subgenomic or defective—the undiluted serial passages with the parental virus are continued. After some passages, the evolution of the RNA pattern is analyzed throughout the serial passages and, for this purpose, cells of the suitable cellular system are infected with viruses originating from different passages and the produced RNAs are analyzed by conventional techniques, for example, metabolic labeling with ³²P_(i) or hybridization with a suitable oligonucleotide. A detailed description of the obtainment and characterization of some defective RNAs derived from TGEV is made in Example 1.

[0041] With the defective RNAs it is possible to obtain the corresponding cDNA—complementing or substantially complementing—the aforesaid defective RNAs, by means of a, reverse transcriptase (RT) reaction and polymerase chain reaction (PCR) amplification, hereinafter RT-PCR. After this, it is possible to clone the cDNA in appropriate plasmids, for example Bluescript II, under the control of efficient promoters. The resulting plasmids contain the defective viral genome under the control of some regulatory elements, containing signals for the regulation and control of replication and for the initiation and termination of transcription and translation. Therefore, these plasmids can include polyA sequences, auto-catalytic cut sequences or for the recognition of restriction enzymes allowing the insertion of the heterologous DNA, and the corresponding regulation, control and termination signals.

[0042] The plasmids thus obtained, containing the defective genome, or the corresponding cDNA, can be manipulated by conventional genetic engineering techniques in order to clone with increased efficiency, at least, one heterologous DNA sequence coding for a specific activity, under the control of the promoter of a gene present in the defective genomes or any other promoter of the virus from which the defective genome or variant of these promoters derives, and of the regulatory sequences contained in the resulting expression vector. Example 2 describes the generation of expression vectors coding for antigens inducing protection against different viruses.

[0043] The expression vectors obtained from this invention can express one or more activities, such as one or more antigens capable of inducing immune responses against different infectious agents, or one or more antibodies providing protection against one or more infectious agents. In one specific and preferred realization, these vectors express at least one antigen capable of inducing a systemic immune response or mucosal immune response against different infectious agents that propagate in respiratory or enteric mucosae. In another specific and preferred realization, the said expression vectors express, at least, one gene coding for the heavy and light chains of an antibody of any isotype (for example: IgG₁, IgA, etc.) that confers protection against an infectious agent. In a specific case the antibody expressed is the monoclonal antibody identified as 6A.C3 (see Example 4) which neutralizes TGEV, expressed with isotypes IgG₁ or IgA in which the constant part of the immunoglobulin is of porcine origin.

[0044] In a specific realization of this invention, cloning of the heterologous genes in a plasmid, containing one cDNA of a defective RNA derived from, was done after ORF1b, following the S gene initiation codon (AUG), and in reading frame with this gene (Example 2). Alternatively, the heterologous DNA sequences can be cloned in other areas of the genome, for example, in the zones corresponding to ORFs 1, 2 or 3 of TGEV. From the resulting plasmids, RNAs were expressed using a suitable polymerase, with which appropriate cells—previously infected with an attenuated helper virus—were transformed, enabling to recover virions containing the helper virus genome and other virions with defective genome (FIG. 17).

[0045] Alternatively, this invention's expression vectors allow the expression of one or several genes using the strategy described above. To that end, one or several promoters can be used, or a promoter and several internal ribosome entry sites (IRES) or, alternatively, several promoters and an internal ribosome entry site.

[0046] This invention also provides a recombinant system to express heterologous proteins comprising (a) the vector described above and (b) a helper virus which facilitates the proteins involved in the replication and encapsidation of the recombinant defective viral genome. Therefore, a recombinant system for the expression of heterologous proteins is provided, based on recombinant defective viral genomes expressing protein with at least one specific activity, comprising:

[0047] a) a recombinant vector containing a defective viral genome for which, in its case, a cDNA has been obtained, capable of being manipulated by means of conventional genetic engineering, containing the viral replicase recognition signals located at the 3′ and 5′ ends, comprising, additionally, internal deletions, and at least one internal DNA sequences coding for one activity, for example, an antigen capable of conferring systemic and mucosal immunity; or an antibody conferring protection against and infectious agent; and

[0048] b) a helper virus providing the functional and structural proteins for the replication and encapsidation of the defective genome.

[0049] Alternatively, the aforesaid recombinant system for the expression of heterologous proteins comprises an expression vector, of the type described above, comprising the complete sequence coding for the parental virus replicase and a helper parental virus which provides structural proteins for the encapsidation of the defective genome and, optionally, the functional proteins (replicase) for the replication of the defective viral genome.

[0050] These systems enable the expression, either of antigens capable of inducing an immune response or of antibodies that confer protection against infectious agents, for which reason they are suitable to be used with vaccinal purposes and for protection against infections.

[0051] This invention also provides vaccines capable of inducing protection against infections caused by various infectious agents in different animal species comprising (i) the recombinant system described above, consisting of: (a) an expression vector based on a defective viral genome in which the heterologous DNA sequence is cloned, and (b) the helper virus collaborating in the replication of the defective genome together with, optionally, (ii) a pharmaceutically acceptable excipient. The vaccines provided by this invention are, therefore, suitable for the conferring of immunity against various infectious agents affecting different animal species.

[0052] “To confer immunity”, in the sense used in this description, should be understood to mean the starting in a receptor organism (animal to be treated), by the recombinant system as described above, of the suitable mechanisms, such as antigen-presenting cells, B and T lymphocytes, antibodies, substances potentiating cell immune response (interleukines, interferons, etc.), cell necrosis factors and similar substances that produce protection in animals against infections caused by pathogenic agents.

[0053] The vaccines provided by this invention can be monovalent or multivalent, depending on whether the expression vectors present in the recombinant system express one or more antigens capable of inducing an immune response against one or more infectious agent. The expression vectors can be monovalent or polyvalent depending on whether they express one or more antibodies conferring protection against one or more infectious agent.

[0054] Species specificity is controlled in such a way that the helper virus will express an envelope protein suitable for recognition by the cell receptors of the corresponding species. A specific group of vaccines obtained from this invention comprise as helper virus a coronavirus, preferably a porcine, canine or feline coronavirus.

[0055] These vaccines are especially indicated against infectious agents that affect the porcine, canine and feline species, infecting the mucosae or using mucosae as the route of entrance.

[0056] A specific realization of this invention provides monovalent vaccines capable of protecting pigs, dogs and cats against various porcine, canine and feline infectious agents, and tropism is controlled by expressing the S glycoprotein of a coronavirus.

[0057] The monovalent vaccines against porcine infectious agents can contain an expression vector expressing an antigen selected from the group composed essentially of antigens of the following porcine pathogens: Actinobacillus pleuropneumoniae, Acfinobacillus suis, Haemophilus parasuis, Porcine parvovirus, Leptospira, Escherchia coli, Erysipelothrix rhusiopathiae, Pasteurella multocida, Bordetella bronchiseptica, Clostridium sp., Serpulina hyodisentedae, Mycoplasma hyopneumoniae, porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus, rotavirus, or against the pathogens that cause porcine reproductive and respiratory syndrome, Aujeszky's disease (pseudorabies), swine influenza, transmissible gastroenteritis and the etiologic agent of atrophic rhinitis and proliferative ileitis.

[0058] The monovalent vaccines against canine infectious agents can contain an expression vector expressing an antigen selected from the group constituted essentially of antigens of the following canine pathogens: canine herpesviruses, canine adenovirus types 1 and 2, canine parvovirus types 1 and 2, canine reovirus, canine rotavirus, canine coronavirus, canine parainfluenza virus, canine influenza virus, distemper virus, rabies virus, retrovirus and canine calicivirus.

[0059] The monovalent vaccines against feline infectious agents can contain an expression vector expressing an antigen selected from the group constituted essentially of antigens of the following feline pathogens: feline calicivirus, feline immunodeficiency virus, feline herpesviruses, feline panleukopenia virus, feline reovirus, feline rotavirus, feline coronavinus, feline infectious peritonitis virus, rabies virus, feline Chlamydia psittaci, and feline leukemia virus.

[0060] The vectors can express an antibody that confers protection against an infectious agent, for example, a porcine, canine or feline infectious agent, such as those described above. In a specific realization, some vectors were produced expressing the recombinant monoclonal antibody identified as 6A.C3 which neutralizes VGPT.

[0061] The vaccines obtained from this invention are capable of protecting piglets by means of inducing lactogenic immunity, which is of special interest in the protection of neonatal piglets against infections of the intestinal tract.

[0062] In general, the vaccines obtained from this invention can contain a quantity of antigen capable of introducing into the animal to be immunized a helper virus a titer of, at least, 10⁸ plaque forming units (pfu).

[0063] As excipient, a diluent such as physiological saline serum or other similar saline solutions may be used. Likewise, these vaccines can also contain an adjuvant like those commonly used in the formulation of vaccines: aqueous (aluminum hydroxide, QuilA, alumina gel suspensions and similar), or oily [based on mineral oils, glycerides and fatty acid by-products and their mixtures, for example, Marcol 52 (ESSO Españiola S.A.), Simulsol 5100 (SEPIC) and Montanide 888 (SEPIC)].

[0064] These vaccines can also contain cell response potentiator substances (CRP), i.e., T helper cell (Th₁ and Th₂) subpopulation potentiator substances, such as interleukin 1 (IL-1), IL-2, IL-4, IL-5, IL-6, IL-12, g-IFN (gamma interferon), cell necrosis factor and similar substances that might, in theory, provoke cell immunity in vaccinated animals. These CRP substances could be used in vaccine formulations with aqueous or oily adjuvants. Other types of adjuvants that modulate and immunostimulate cell response can also be used, such as MDP (muramyl dipeptide), ISCOM (immuno stimulant complex) or liposomes.

[0065] This invention provides multivalent vaccines capable of preventing and protecting animals from different infections. These multivalent vaccines can be prepared from expression vectors in which the different sequences coding for the corresponding antigens have been inserted in the same recombinant vector, or by constructing independent recombinant vectors that will be mixed afterwards for their co-inoculation together with the helper virus. Therefore, these multivalent vaccines comprise a recombinant system in which the expression vector itself contains more than one DNA sequence coding for more than one infectious agent or, alternatively, the recombinant system used in the preparation of the vaccine can contain different expression vectors, each one of which expresses at least one different antigen. The actual limitation in this type of multivalent vaccines is that the mentioned expression vectors must express antigens of infectious agents of the same animal species and that the helper virus must be the suitable one for that particular species. Analogously, multivalent vaccines can be prepared that comprise multivalent vectors using sequences coding for antibodies that confer protection against infectious agents instead of sequences coding for the antigens. These vectors can contain a recombinant system comprising either an expression vector containing more than one DNA sequence coding for more than one antibody or different expression vectors expressing, each one of them, at least, one different antibody.

[0066] A specific realization of this invention provides vaccines capable of protecting pigs, dogs and cats against various porcine, canine and feline infectious agents, respectively. To that end, the expression vectors contained in the recombinant system of the vaccine must express different antigens of the mentioned porcine, canine or feline pathogens.

[0067] The vaccines of this invention can be presented in liquid or freeze-dried form and can be prepared by suspending the recombinant systems in the excipient. If the said systems are in freeze-dried form, the excipient itself can be the diluent.

[0068] Alternatively, the vaccines attained by means of this invention can be used in combination with other conventional vaccines, either as part of them or as diluent or freeze-drying fraction to be diluted with other, either conventional or recombinant, vaccines.

[0069] The vaccines provided by this invention can be administered to the animal via oral, nasal, subcutaneous, intradermal, intraperitoneal or intramuscular routes, or by aerosol.

[0070] The invention also provides a method for the immunization of animals, especially pigs, dogs and cats, against various infectious agents simultaneously—comprising the administration via oral, nasal, subcutaneous, intradermal, intraperitoneal or intramuscular routes, or by aerosol (or their combined forms)—of a vaccine containing an immunologically efficacious quantity of a recombinant system provided by the present invention.

[0071] Additionally, the invention also provides a method for the protection of newborn animals against infectious agents that infect the said animals, consisting of the administration via oral, nasal, subcutaneous, intradermal, intraperitoneal and intramuscular routes, or by aerosol (or their combined forms), to mothers, before or during the period of gestation, or to their progeny, of a vaccine containing an immunologically efficacious quantity of a recombinant system provided by this invention.

[0072] The invention is illustrated by means of the following examples that describe in detail the obtainment of defective viral genomes, their characterization, the construction of plasmids and their manipulation with the purpose of obtaining the expression vectors and the induction of neutralizing antibodies against different infectious agents of various species.

EXAMPLE 1

[0073] Generation of Defective Particles Derived from TGEV

[0074] 1.1 Undiluted Serial Passages of TGEV Strains at High Multiplicity of Infection (m.o.i.)

[0075] In order to promote the generation of defective particles, or the imposition of those already exisiting in small proportion in the viral population, serial passages of different undiluted TGEV isolates were made in different cell systems. Because the frequency with which DI particles are generated can vary much in different virus-cell systems, the passages were done with different TGEV isolates (THER-1 and PUR46-mar 1CC12) in ST (swine testis, swine testicle epithelial cells) cell lines.

[0076] Strain THER-1 (transmissible gastroenteritis Helper Enteric and Respiratory coronavirus, strain 1) is a mutant attenuated by 20 passages in ST cell cultures derived from the PUR46-MAD strain [Sánchez et al., Virology 174, 410-417 (1990)]. Strain PUR46mar 1CC12 is also described by Sanchez et al (supra).

[0077] Each TGEV strain was passaged undiluted 35 times in ST cells. The m.o.i. of the first passage in each one of the three cases was 100 pfu per cell. The supernatant of each passage was collected between 20 and 48 hours post infection (h.p.i.), upon observance of a clear cytopathic effect—normally when the said effect was affecting more than half of the cell monolayer—, and half of the volume of this supernatant was used in the infection of the next passage. Virus titer variation with passage number is shown in FIG. 4. Viral titer ranged between two logarithmic units throughout the serial passages of each virus. In the case of strain THER-1, the titer in passages 30 to 46 was lower than in the first thirty passages.

[0078] The viruses that had been passaged 35 times in ST cells were used for the analysis of intracellular RNAs produced in the infection. The RNAs, metabolically labeled with ³²P₁ between hours 6 and 9 post infection, were analyzed [Maniatis et al., Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, (1982)]. In THER-1-p35 infection (THER-1 strain virus passaged 35 times at high m.o.i.) three intense bands were observed which did not correspond to any viral mRNA. These bands were located between the genomic RNA and the S gene messenger (FIG. 5). To analyze the nature of these new subgenomic RNAs, undiluted serial passages were continued with strain THER-1. After 46 passages, the evolution of the RNA pattern throughout the serial passages was analyzed. To that end, ST cells were infected with virus from various passages and the produced RNAs, metabolically labeled, were analyzed in a denaturing agarose gel (FIG. 5). Whereas only genomic RNAs and subgenomic viral messengers were detected in the first passages, in passage 30 three new RNAs of 22, 10.6 and 9.7 kb (RNAs A, B and C shown in FIG. 5 as DI-A, DI-B and DI-C, respectively) were detected. These subgenomic RNAs remained in stable form throughout the following 15 passages, interfering notably with genomic RNA replication and the synthesis of the mRNAs of the helper virus (FIG. 5, lanes 30 to 45). These results indicate that the three RNAs generated or amplified by the undiluted serial passages are stable and that at least one of them is interfering.

[0079] 1.2 Characterization of Subgenomic RNAs

[0080] 1.2.1 Analysis of the Ends and Internal Regions

[0081] To determine if subgenomic RNAs A, B and C had the standard structure of a coronavirus defective RNA, and especially, if they preserved the 5′ and 3′ ends of the wild genome and that its small size was due to internal deletions, several hybridization assays were carried out with probes specific for the virus. To that end, the RNA from cells infected with the THER-1-STp35 virus (THER-1 strain viruses, passaged 35 times in ST cells) was extracted and its hybridization was analyzed with specific viral probes in a Northern-blot assay (Maniatis et al., supra) using oligonucleotides complementary to the leader and the viral 3′ end sequence. In each case, the RNA from cells infected with THER-1-p1 virus (strain THER-1 virus passaged once in ST cells) and from uninfected ST cells (NI) (lanes 1 and 2 of each filter, respectively) were used as control. The oligonucleotides used as probes are complementary to the leader RNA (positions 66-91 of the 5′ end of the parental genome), to the untranslated region of the 3′ end (nucleotides 28524-28543 of the 5′ end of the parental genome), and to structural genes M and N (positions 97-116 and 5-24 starting from the primer AUG of each gene, respectively). The bars on the right indicate the positions of the viral mRNAs and subgenomic RNAs A, B and C.

[0082] As shown in FIG. 6, the two oligonucleotides hybridized with all the parental virus mRNAs, and they also detected RNAs A, B and C, indicating that these RNAs have gone through internal deletions and maintained their ends. As an initial approach to an analysis in regard to which genomic sequences were present in these RNAs, the RNA of infected cells was hybridized with oligonucleotides complementary to the genes of structural viral proteins S, M and N. None of them hybridized with the defective RNAs, suggesting that the structural protein genes had been deleted. Thus, subgenomic RNAs A, B and C maintain the parental virus ends and have internal deletions.

[0083] 1.2.2 Propagation of RNAs A, B and C

[0084] In order to verify that RNAs A, B and C are defective genomes, dependent on the parental virus for their propagation in culture, ST cells were infected with virus THER-1-STp41 (THER-1 virus strain passaged 41 times in ST cells) at different m.o.i.: 10, 0.1, 0.01 and 0.001 pfu/cell. The virus resulting from this passage, collected at 10 h.p.i., was titrated and amplified in a second passage in ST cells, which in turn was used to infect new cells from which the cytoplasmic RNA was extracted (Maniatis et al., supra). The RNA was analyzed in a Northern-blot assay using an oligonucleotide complementary to the leader RNA. FIG. 7 shows the results obtained. The m.o.i. are indicated over each lane (10⁻³, 10⁻², 10⁻¹ and 10 pfu/cell). As negative control was included the RNA from an infection of ST cells with THER-1-p1 virus, which does not contain subgenomic RNAs, at m.o.i. of 10 pfu/cell (first lane). In the lane corresponding to the infection with THER-1-STp41 virus at m.o.i. of 10 pfu/cell, positive control, the genomic RNAs (mRNA 1) are labeled, and so are defective RNAs A, B and C (represented as DI-A, DI-B, DI-C) and the corresponding S gene (mRNA 2).

[0085] It is observed that when the m.o.i. of the first passage (the “bottleneck” in this experiment, as the passages that follow are amplification passages) is 0.1 pfu/cell or less, RNAs A, B and C are lost, in conditions in which the genomic RNA and the mRNA of the virus are detected in the expected proportions (FIG. 7). The three defective RNAs are maintained when the m.o.i. is 10 pfu/cell. Because RNAs A, B and C are found in higher proportion than genomic RNA in the THER-1-p41 virus used in the infection, these results show that the replication or propagation of these RNAs requires that the cells be infected by defective virus and also by the helper virus. Thus, RNAs A, B and C require functions of the helper virus which are to be provided in trans. Therefore, RNAs A, B and C are defective genomes, depending on a helper virus for their propagation.

[0086] 1.2.3 In vito Generation, Amplification, Propagation and Interference of DI RNA in Another Cell Line

[0087] In vitro generation, amplification, propagation and interference of RNA DI are specific to the cell line, and for this reason the effect that a change of cell line might have on defective RNAs was studied. To that end, the THER-1-STp46 virus (THER-1 virus passaged 46 times at a high m.o.i. in ST cells) was subjected to a new series of undiluted passages, in intestinal porcine epithelial cells (IPEC) and porcine macrophages (PM). FIG. 8 shows titer variation and passage number through 10 passages in IPEC (FIG. 8A) and 5 passages in PM (FIG. 8B). Viral yield in both cell lines was lower than what was obtained in ST cells, and the estimate is that there was a variation ranging between 20 and 0.2 pfu/cell in the m.o.i. of each passage.

[0088] The RNA produced in ST cells infected with THER-1-STp46-IPECp1 (THER-1-STp46 virus passaged one time in IPEC cells) and THER-1-STp46-IPECp10 (THER-1-STp46 virus passaged 10 times in IPEC cells) was labeled with ³²P_(i) and analyzed in a denaturant agarose gel (FIG. 8C).

[0089] The RNA of ST cells infected with THER-1-STp46PMp1 virus (THER-1-Tp46 virus passaged one time in PM cells) and THER-1-STp46-PMp5 (THER-1-STp46 virus passaged 5 times in PM cells) was analyzed in a Northern-blot assay with an oligonucleotide complementary to the leader RNA (FIG. 8C).

[0090] The results appear on FIG. 8C, which shows that the three defective RNAs remained in the first passage in both cell lines, but only RNA A persisted throughout, at least, five passages in PM, and ten passages in IPEC. In both cases the positions of the RNAs corresponding to the wild genome (1) are marked, RNAs A, B and C (DI-A, DI-B and DI-C respectively) and mRNA 2 (protein S). In the lane corresponding to the THER-1-STp46-PMp5 virus RNA, the position of genomic RNA is indicated—observed only when the exposition period of the autoradiograph was tenfold the period shown on FIG. 8C.

[0091] 1.3 Encapsidation of Defective Genomes

[0092] In order to study whether defective RNAs have the capacity to encapsidate, a partial purification was done simultaneously on viruses THER-1-STp1 (THER-1 virus passaged one time in ST cells) and THER-1-STp41 (THER-1 virus passaged 41 times in ST cells) by means of centrifugation through a 15% weight/volume (w/v) sucrose cushion. The RNA was extracted from the purified virions, and analyzed in agarose gel by means of ethidium bromide staining (FIG. 9A). In passage 41 virions, RNAs A, B and C were detected with the same intensity as genomic RNA, indicating that the three defective RNAs encapsidate efficiently.

[0093] To determine if the defective genomes coencapsidate with the complete genome, or if, on the contrary, they encapsidate independently, THER-1-STp41 virus was purified by centrifugation through sucrose cushions of varying densities, or through continuous sucrose gradients. The RNA of the purified virions in each case was analyzed in a Northern-blot assay with an oligonucleotide complementary to the leader RNA (FIG. 9B). When centrifugation was done through a sucrose cushion of 31% (w/v) (d=1.19 g/ml), only wild genome was detected in the sedimented virions. However, when a sucrose cushion of lower density, 15% (w/v) (d=1.11 g/ml), was used, the three defective RNAs were detected, in addition to the complete genome. In a continuous sucrose cushion (15-42%, w/v) it was possible to enrich the defective virions in the upper fractions of the gradient (density close to 1.15 g/ml), and the standard virions in the lower fractions (density close to 1.20 g/ml), as shown on lanes d and e of FIG. 9B. The upper band in each lane corresponds to the wild type genomes and to defective genome A (DI-A), and the lower band corresponds to defective genomes B and C (DI-B and DI-C). These results indicate that RNAs A, B and C encapsidate efficiently, and that genomes DI-B and DI-C (10.6 and 9.7 kb) do so independently from the wild type genome, in defective virions that are lighter than standard virions.

[0094] 1.4 Cloning and Sequencing of Defective RNAs B and C. Determination of Their Primary Structure.

[0095] 1.4.1 Synthesis of Complementary DNA and Amplification of RNAs B and C.

[0096] The size of defective RNAs B and C had been estimated, based on their mobility in electrophoresis gels, in 10.6 and 9.7 kb, respectively. On account of their large size, it was not possible to amplify the defective RNAs in a single reverse transcriptase and polymerase chain reaction (RT-PCR) using primers complementary to genome ends. In order to overcome this limitation, the defective genomes were amplified in four independent reactions, using pairs of primers allowing four overlapping fragments to cover the total genome length in every case. These overlapping fragments were designated as a, b, c and d, arranged from the 5′ end to the 3′ end (FIG. 10). The THER-1-STp41 virus RNA extracted from purified virions was used as a template. This RNA contained the three defective RNAs A, B and C, in addition to the parental genome. As a control, an amplification of the genomic RNA of the THER-1 wild virus was carried out simultaneously.

[0097] The sequence and position of the oligonucleotides used as primers in the RT-PCR reaction is indicated in Table 2. TABLE 2 Characteristics of the oligonucleotides used as primers in the RT-PCR reactions SEC. ID. ORF Position in the Restriction No. Polarity Coronavirus RNA DI-C^(b)) Site 1 + Leader TGEV 15-41 — 2 − ORF1a FIPV 1874-1887 — 3^(a)) + ORF1a TGEV 1524-1550 Xbal 4^(a)) − ORF1b TGEV 4365-4389 Xbal 5^(a)) + ORF1b HCV229E 4097-4114 EcoRl 6^(a)) − ORF1b TGEV 7633-7650 EcoRl 7 + ORF1b TGEV 7633-7650 — 8^(a)) − 3′ UTR TGEV 9691-9707 Spel 9 + ORF1b TGEV 8251-8270 —

[0098] The amplification by RT-PCR with primers 1 and 2, of the THER-1-STp41 virus RNA and the parental virus THER RNA gave rise to a dominant PCR product of 1.9 kb (FIG. 11, fragment a). The minor bands observed in this reaction are due to unspecific hybridizations, as they appear in the two lanes. The same RT-PCR reaction was performed starting from an agarose fraction containing a pool of DI-B and DI-C RNAs extracted from a purification with gel, with the same results. This indicates that fragment a is common to all RNA DIs, and corresponds to the 1.9 kb region of the 5′ end of the wild TGEV genome.

[0099] The amplification with oligonucleotides 3 and 4 gave rise to a unique PCR 2.8 kb product starting from the THER-1-STp41 virus RNA (FIG. 11, fragment b). No PCR product was obtained from the THER-1 control virus RNA, as the size of the expected product was 12 kb. Based on these data, it can be deduced that at least one defective genome has one b fragment of 2.8 kb, and that the others have the same fragment or a larger one which is not detectable in PCR reactions due to its large size.

[0100] Oligonucleotides 5 and 6, separated by 4.6 kb in the parental genome, gave rise to two different products of 3.5 and 4.6 kb from the THER-1-STp41 virus RNA (FIG. 11, fragment c). The 4.6 kb product was also obtained from the wild virus RNA used as control. These results suggest that fragment c contains one deletion in at least one defective genome (probably in DI-C, the most abundant defective genome), giving rise to a 3.5 kb fragment by PCR. The 4.6 kb fragment derives from the parental genome present in the THER-1-STp41 virus RNA population, and from those defective genomes that have maintained this region of the genome.

[0101] The amplification by RT-PCR with primers 7 and 8 of the parental virus genomic RNA did not generate any bands (FIG. 11, fragment d), since the separation between these oligonucleotides is 9.5 kb in the complete genome (FIG. 10). In contrast, two very intense bands of 1.9 and 2.1 kb were observed when the THER-1-STp41 virus RNA was used as template. These bands appear as a broad continuous band, corresponding to fragment d (FIG. 11), probably because they co-migrate with a group of minor bands around the 1.9 kb band, preventing their resolution in gels. Heterogeneity has been observed in the size of cloning fragment d (see below).

[0102] 1.4.2 Assigning Amplification Products (a, b, c and d) to the Different Defective RNAs.

[0103] In order to assign the d fragments of sizes varying between 1.9 and 2.1 kb to the different defective genomes, the THER-1-STp41 virus RNA, which had been used as a template, was fractioned in an agarose gel until a clear separation was achieved of the bands corresponding to the RNAs of the wild genome, DI-A, DI-B and DI-C. The bands corresponding to each one of these four RNAs were cut independently and used as a template in the amplification reaction of RT-PCR with oligonucleotides 8 and 9. Starting from the purified genomic band RNA, no PCR product was obtained. From RNA DI-B, a predominant 1.9 kb PCR product was obtained, although less abundant DNAs were also obtained, of variable size close to 1.9 kb, which indicates a certain heterogeneity in this zone. The amplification of RNA DI-C gave rise to a dominant 2.1 kb PCR product. These results allowed to assign the 1.9 kb fragment to the defective RNA B, and the 2.1 kb fragment to RNA DI-C.

[0104] Once the d fragments had been assigned, the 3.5 and 4.6 kb c fragments obtained with primers 5 and 6 were assigned to defective RNAs C and B, respectively, since the sum of fragments a to d resulting from this assignment coincided in each case with the sizes of RNAs B and C, estimated by mobility.

[0105] Once the complete sequence of genomes B and C had been determined, fragment assignment was verified by amplifying each purified band RNA, using oligonucleotides flanking specific deletions. The assignment of the fragments was also confirmed by Northern-blot assay, using oligonucleotides mapping the DI-B regions that were not present in DI-C, and vice versa.

[0106] 1.4.3 Cloning and Sequencing the Overlapping Fragments a, b, c, and d.

[0107] The four overlapping DNA fragments a (1.9 kb), b (2.8 kb), c (3.5 kb) and d (2.1 kb), complementary to RNA C, were cloned in Bluescript SK⁻. At least two clones derived from independent RT-PCR reactions were sequenced. The sequence of the positions that did not coincide in the different clones (possibly errors of the Taq polymerase) were sequenced directly from the corresponding uncloned PCR products. Following this procedure, the RNA DI-C consensus sequence was determined. An average of 1 error of Taq polymerase was obtained each copied 1.2 kb. The complete sequence of the DI-C genome is shown on FIG. 12.

[0108] The complete RNA DI-C sequence obtained this way was compared with the ORFs 1a and 1b sequence of the PUR46-PAR virus [Eleouet et al., Virology 206, 817-822 (1995)], and with the sequence determined in our laboratory for the other THER-1 virus ORFs. In the complete RNA DI-C sequence, 14 nucleotide differences were found in comparison with the sequence of strain PUR46-PAR. These positions were sequenced in strain THER-1, the parental virus of the defective genome, in order to define the specific changes of the genomic defective RNA DI-C. The DI-C RNA sequence only presented three nucleotide differences in comparison with the corresponding sequence of the parental virus, and one insertion in position 9189, which does not affect any ORF (FIG. 12).

[0109] 1.4.4 Primary DI-C and DI-B Genome Structures.

[0110] The sequence data indicated that the DI-C genome was composed of four discontinuous parental genome regions (FIG. 13) comprising: a) the 2144 nucleotides of the 5′ end of the genome; b) 4540 nucleotides corresponding to the region between positions 12195 and 16734 of the parental genome, which includes the overlapping area between ORFs 1a and 1b, and approximately the 5′ half of ORF 1b; c) a region of 2531 nucleotides corresponding to positions 17843-20372 of the wild genome, and which comprises the 3′ half of ORF1b and the first 8 gene S nucleotides; and d) the 493 nucleotides of the 3′ end.

[0111] The primary structure of the DI-B genome was determined by sequencing of cloned fragments a and b (common to genome DI-C), c (like the parental genome) and d (specific to genome DI-B). Genome DI-B is composed of three discontinuous regions of the genome (FIG. 14): a) the 2144 nucleotides of the 5′ end of the genome, common to all DI-B clones, and identical to region I of RNA DI-C; b) a region variable in size, of 8178-8243 nucleotides corresponding to positions 12195-20369 to 20436 of the parental genome, and which includes the overlapping zone between the two ORFs of gene 1, the complete ORF1b, and the first nucleotides of gene S; and c) nucleotides 278 to 303 of the 3′ region of the genome.

[0112] The clones constituting the population designated as genomes DI-B differ in the size of the deletion that took place between regions II and III, which starts at the beginning of gene S (between nucleotides 6 and 73) and finishing at the end of gene 7 (between nucleotides 195 and 233).

[0113] The 5′ end sequence of parental RNA THER-1 was determined by direct sequencing of the RNA, and is 5′-NCUUUUAAAG-3′. The nature of the first “N” nucleotide of the sequence has not been determined. So far, the sequence of the 5′ end of three TGEV isolates, PUR46-PAR, PUR46-BRI and FS772/70, has been described [Eleouet et al., supra; Page et al., Virus Genes 4, 289-301 (1990); Sethna et al., J. Virol. 65, 320-325 (1991)] and they all differ in the first nucleotide. The sequence of the defective RNAs leader must be the same as that of the parental virus leader, in view of the interchange of leaders that takes place in a coronavirus infection [Makino et al., J. Virol. 57, 729-737 (1986)].

[0114] The three defective RNAs contain polyA, as they join themselves to oligo dT columns (results not shown).

[0115] 1.4.5 RNAs B and C Keep the Overlapping Region between ORFs 1a and 1b, which Includes the Motive Responsible for the Translocation (−1) of the Ribosome

[0116] In accordance with the sequences assigned to genomes DI-C and DI-B, it is possible to predict ORFs of 6370 and 10003 nucleotides, respectively, starting at nucleotide 315, counting from the 5′ end of the genome. The RNA DIC ORF ends at the termination codon generated at the joint site of discontinuous regions II and III, where the internal deletion had taken place in ORF 1b, at position 6685 of the DI-C genome. The DI-B genome ORF ends at the natural termination codon of ORF1b.

[0117] The two defective RNAs keep the overlapping zone between ORFs 1a and 1b, which includes the sliding sequence and the tertiary structure motive pseudoknot, responsible for the translocation (−1) of the ribosome in this zone (Eleouet et al., supra). FIG. 15 shows the possible RNA secondary and tertiary structures in this zone. The structure proposed by Eleouet et al. for the pseudoknot in this zone, is the one indicated in C and D; however, there are other possible structures (as indicated in A and B), although it is not know which is the correct one.

[0118] A description has been made, indicating that the translocation occurs with an efficiency of 20% in TGEV (Eleouet et al., supra) and enables the continuous translation of gene 1. The fact that RNAs DI-B and DI-C (and probably DI-A RNA) keep this region of the parental genome suggests that it could be necessary for RNA replication or for genome propagation.

[0119] There are two other small ORFs in defective genomes DI-C and DI-B. One of them, previous to the long reading frame, codes for a peptide of three amino acids, which is also found in the wild type genome, whose function is unknown. The other ORF starts in both cases in the AUG of gene S, and codes for a peptide of 16 amino acids in DI-C, and a peptide of variable size in DI-B. It is unknown whether these ORFs are functional. The only two transcription promoter consensus sequences (CUAAAC) of the virus that are kept are precisely those preceding gene 1 and gene S, in defective RNAs B and C. These sequences are marked in FIG. 12.

[0120]FIG. 16 shows the mapping of RNAs A, B and C by hybridization with oligonucleotides specific to the virus in Northern-blot assays. The THER-1-STp41 virus RNA was fractioned in agarose gels until a clear separation of the RNAs from the parental genome and DI, A, B and C had been obtained. The RNA was transferred to nylon filters which hybridized with various oligonucleotides marked with ³²P_(i), hybridized with the parental genome (+), and hybridized (+) or not (−) with the defective genomes. The approximate locations of the sequences complementary to the oligonucleotides in the parental genome are shown marked with arrows. Their exact sequence and position appear in Table 3. All the oligonucleotides hybridized with the parental genome, and the expected results with RNAs B and C were obtained. TABLE 3 Position in the ON ID Sec. No. Polarity Gene in TGEV genome^(a)) 1 10 − Leader 66-91 2 11 − ORF1a 2151-2170 3 12 − ORF1a 6121-6140 4 13 − ORF1a 8684-8703 5 14 − ORF1a 12261-12280 6 15 − ORF1b 14148-14167 7 16 − ORF1b 17363-17381 8 17 − ORF1b 18792-18811 9 18 − gene S 1055-1074 10 19 − gene S 1980-1999 11 20 − gene S 3600-3619 12 21 − gene M  97-116 13 22 − gene N  5-24 14 23 − UTR-3′ 28524-28543

EXAMPLE 2

[0121] Generation of Expression Vectors

[0122] The cDNA coding for RNA DI-C has been cloned in a Bluescript II plasmid, under the control of the phage T7 promoter. This cDNA includes polyA sequences, a hepatitis delta virus (HDV) ribozyme, and phage T7 termination signals. One of these plasmids, whose construction appears in FIG. 17, has been denominated pDIC-1. These plasmids can be manipulated to clone in them heterologous genes under the control of the gene S promoter present in the defective genome, or another TGEV promoter, or a variant of them with increased efficiency.

[0123] The cloning of these heterologous genes was done after ORF1b, following the S gene initiation codon (AUG), and in reading phase with this gene.

[0124] From these cDNAs, RNAs were expressed using the phage T7 polymerase, with which ST cells that had been infected previously with the attenuated helper virus THER-1 were transformed, enabling to recover virions, containing the helper virus genome, and other virions, containing the corresponding defective genome. These viruses, freeze-dried in presence of 2% fetal calf serum, were used as vaccine for the induction of specific antibodies against agents that infect the gastrointestinal and respiratory tracts of pigs, dogs and rabbits.

[0125] The tropism of the vectors was specifically targeted to the porcine, canine, or feline species, using the appropriate attenuated helper viruses.

EXAMPLE 3

[0126] Induction of Neutralizing Antibodies

[0127] 3.1 Induction of Protection Against Porcine Epidemic Diarrhea Coronavirus (PEDV)

[0128] Pigs were immunized using a recombinant system consisting of helper virus (THER-1), and the pDIC-1 plasmid in which the PEDV glycoprotein S gene had been cloned.

[0129] The immunizations were done by administering 10⁹ pfu per piglet, via oral route.

[0130] Presence of neutralizing antibodies was assayed in the sera of animals vaccinated at 15, 30, 45 and 60 days post immunization; and presence of antibodies specific to PEDV was determined using a radio immuno-assay (RIA) (Maniatis et al., supra).

[0131] With the sera collected on the 45th day post immunization, total protection was provided against infection by PEDV (strain SEG86-1) in 10-day-old piglets, when these sera had been pre-incubated with the virulent virus before oral administration.

[0132] 3.2 Induction of Protection Against Canine Coronavirus

[0133] Dogs were immunized using a recombinant system consisting of helper virus (canine coronavirus strain Fort Dodge), and the pDIC-1 plasmid in which the canine coronavirus glycoprotein S gene had been cloned (strain Fort Dodge).

[0134] The immunizations were done by administering 10⁹ pfu per dog via oral route.

[0135] Presence of neutralizing antibodies in the sera of the animals vaccinated at 15, 30, 45 and 60 days post immunization was assayed, and presence of antibodies specific to canine coronavirus was determined using RIA.

[0136] With the sera collected on the 45th day post immunization, total protection was conferred against infection by canine coronavirus (strain Fort Dodge) in 10-day old puppies, when these sera were pre-incubated with the virulent virus before oral administration.

[0137] 3.3 Induction of Protection Against Infections Caused By the Arterivirus PRRSV

[0138] Pigs were immunized using a recombinant system consisting of helper virus (THER-1), and pDIC-1 plasmid in which the arterivirus PRRSV ORF3 and ORF5 (strain Fort Dodge) had been cloned.

[0139] The immunizations were done by administering 10⁹ pfu per piglet, via oral route.

[0140] Presence of neutralizing antibodies was analyzed in the sera of the animals vaccinated at 15, 30, 45 and 60 days post immunization, and presence of antibodies specific to PRRSV was determined by RIA.

[0141] With the sera collected on the 45th day post immunization, total protection was provided against infection by PRRSV (strain Fort Dodge) in 10-day-old piglets, when these sera had been pre-incubated with the virulent virus before the administration via oral route.

EXAMPLE 4

[0142] Generation of Expression Vectors

[0143] Following a procedure similar to the one described in Example 2, a cDNA coding for an selfreplicative RNA has been cloned in a Bluescript II plasmid, under the control of the phage T7 promoter. This cDNA includes polyA sequences, a ribozyme of hepatitis delta virus (HDV) and the phage T7 termination signals. One of these plasmids, whose construction is shown in FIG. 18 has been denominated pDIA-6A.C3. This plasmid contains the gene coding for monoclonal antibody 6A.C3 which neutralizes TGEV. The characteristics of monoclonal antibody 6A.C3 and its construction are described In Dr. Joaquin Castilla Carrión's Doctoral Thesis, entitled “Construcción de animales transgeńicos secretores de anticuerpos neutralizantes para coronavirus”, Universidad Autónoma, Madrid, Faculty of Science, December 1996, pages 43-52, 65-79.

[0144] The cloning of the heterologous gene was done after ORF 1b, following the gene S initiation codon (AUG), and in reading frame with this gene.

[0145] Starting from this cDNA, RNAs were expressed using phage T7 polymerase, with which were transformed ST cells previously infected with the attenuated helper virus THER-1, resulting in the recovery of virions containing the helper virus genome and other virions with the corresponding defective genome. These viruses, freeze-dried in the presence of 2% fetal calf serum can be used as vectors for the expression of the recombinant 6A.C3 monoclonal antibody. The tropism of the vectors was made specifically for the porcine species using the appropriate attenuated helper virus.

EXAMPLE 5

[0146] Expression of Neutralizing Antibodies

[0147] Pigs were immunized using a recombinant system constituted of helper virus (THER-1), and the pDIA-6A.C3 plasmid (Example 4) containing the sequence coding for recombinant monoclonal antibody 6A.C3 which neutralizes TGEV.

[0148] The immunizations were done by administering 10⁹ pfu per piglet via oral route.

[0149] Presence of neutralizing 6A.C3 antibodies was assayed in the serum of animals vaccinated at 15, 30, 45 and 60 days post immunization using an RIA [Maniatis et al, supra]. The recombinant antibodies had RIA titers higher than 10³ and are able to reduce the titer of the infectious virus more than 10⁴ fold.

[0150] Deposit of Microorganisms

[0151] The plasmid denominated pDIC-1, introduced in a DH-5 bacterium derived from E. coli, [DH5/pDIC-1], was deposited on Mar. 6, 1996 at the European Collection of Animal Cell Cultures (ECACC), Porton Down, Salisbury, Willshire SP4 OJG, United Kingdom, with corresponding accession number P96030641.

[0152] Additionally, the attenuated helper virus denominated THER-1 was deposited at ECACC on Mar. 6, 1996, with corresponding accession number V96030642.

1 32 1 27 DNA Transmissible gastroenteritis coronavirus 1 gtgagtgtag cgtggctata tctcttc 27 2 21 DNA Transmissible gastroenteritis coronavirus 2 ccgttgtggt gtcacattaa c 21 3 32 DNA Transmissible gastroenteritis coronavirus 3 gcctctagag gagctttgtg gttcacttac ac 32 4 32 DNA Transmissible gastroenteritis coronavirus 4 gctctagagc gtttgaatca acccccaaaa gc 32 5 26 DNA Transmissible gastroenteritis coronavirus 5 ggaattccgg gactatccta agtgtg 26 6 25 DNA Transmissible gastroenteritis coronavirus 6 ggaattccag caatactatt atcaa 25 7 20 DNA Transmissible gastroenteritis coronavirus 7 ttgataatag tattgctggc 20 8 23 DNA Transmissible gastroenteritis coronavirus 8 ggactagtat cactatcaaa agg 23 9 20 DNA Transmissible gastroenteritis coronavirus 9 gatggatgtt gtggtgtgag 20 10 26 DNA Transmissible gastroenteritis coronavirus 10 cgagttggtg tccgaagaca aaatct 26 11 20 DNA Transmissible gastroenteritis coronavirus 11 atacgagcat caatatcacc 20 12 20 DNA Transmissible gastroenteritis coronavirus 12 agagttgcca cagactgcag 20 13 19 DNA Transmissible gastroenteritis coronavirus 13 cagcagttca aagttaccc 19 14 20 DNA Transmissible gastroenteritis coronavirus 14 ccattgttaa gccaacaacc 20 15 20 DNA Transmissible gastroenteritis coronavirus 15 atcacactta ggatagtccc 20 16 19 DNA Transmissible gastroenteritis coronavirus 16 gtctaacaat gtgccaagg 19 17 20 DNA Transmissible gastroenteritis coronavirus 17 gccagcaata ctattatcaa 20 18 20 DNA Transmissible gastroenteritis coronavirus 18 cactgtggca cccttacctg 20 19 20 DNA Transmissible gastroenteritis coronavirus 19 gtacacccac tatgttgtct 20 20 20 DNA Transmissible gastroenteritis coronavirus 20 ttgcgagtga aaacaaatgt 20 21 20 DNA Transmissible gastroenteritis coronavirus 21 ctcacaatca gacgctgtac 20 22 20 DNA Transmissible gastroenteritis coronavirus 22 gacacgttgt ccctggttgg 20 23 20 DNA Transmissible gastroenteritis coronavirus 23 acattttaaa caatcactag 20 24 9714 DNA Transmissible gastroenteritis coronavirus CDS (315)..(2315) modified_base (1) a, t, c, g, other or unknown 24 ncttttaaag taaagtgagt gtagcgtggc tatatctctt cttttacttt aactagcctt 60 gtgctagatt ttgtcttcgg acaccaactc gaactaaacg aaatatttgt ctttctatga 120 aatcatagag gacaagcgtt gattatttcc attcagtttg gcaatcactc cttggaacgg 180 ggttgagcga acggtgcagt agggttccgt ccctatttcg taagtcgcct agtagtagcg 240 agtgcggttc cgcccgtaca acgttgggta gaccgggttc cgtcctgtga tctccctcgc 300 cggccgccag gaga atg agt tcc aaa caa ttc aag atc ctt gtt aat gag 350 Met Ser Ser Lys Gln Phe Lys Ile Leu Val Asn Glu 1 5 10 gac tat caa gtc aac gtg cct agt ctt cct att cgt gac gtg tta cag 398 Asp Tyr Gln Val Asn Val Pro Ser Leu Pro Ile Arg Asp Val Leu Gln 15 20 25 gaa att aag tac tgc tac cgt aat gga ttt gag ggc tat gtt ttc gta 446 Glu Ile Lys Tyr Cys Tyr Arg Asn Gly Phe Glu Gly Tyr Val Phe Val 30 35 40 cca gaa tac tgt cgt gac cta gtt gat tgc gat cgt aag gat cac tac 494 Pro Glu Tyr Cys Arg Asp Leu Val Asp Cys Asp Arg Lys Asp His Tyr 45 50 55 60 gtc att ggt gtt ctt ggt aac gga gta agt gat ctt aaa cct gtt ctt 542 Val Ile Gly Val Leu Gly Asn Gly Val Ser Asp Leu Lys Pro Val Leu 65 70 75 ctt acc gaa ccc tcc gtc atg ttg caa ggc ttt att gtt aga gct aac 590 Leu Thr Glu Pro Ser Val Met Leu Gln Gly Phe Ile Val Arg Ala Asn 80 85 90 tgc aat ggc gtt ctt gag gac ttt gac ctt aaa att gct cgc act gkc 638 Cys Asn Gly Val Leu Glu Asp Phe Asp Leu Lys Ile Ala Arg Thr Xaa 95 100 105 aga ggt gcc ata tat gtt gat caa tac atg tgt ggt gct gat gga aaa 686 Arg Gly Ala Ile Tyr Val Asp Gln Tyr Met Cys Gly Ala Asp Gly Lys 110 115 120 cca gtc att gaa ggc gat ttt aag gac tac ttc ggt gat gaa gac atc 734 Pro Val Ile Glu Gly Asp Phe Lys Asp Tyr Phe Gly Asp Glu Asp Ile 125 130 135 140 att gaa ttt gaa gga gag gag tac cat tgc gct tgg aca act gtg cgc 782 Ile Glu Phe Glu Gly Glu Glu Tyr His Cys Ala Trp Thr Thr Val Arg 145 150 155 gat gag aaa ccg ctg aat cag caa act ctc ttt acc att cag gaa atc 830 Asp Glu Lys Pro Leu Asn Gln Gln Thr Leu Phe Thr Ile Gln Glu Ile 160 165 170 caa tac aat ctg gac att cct cat aaa ttg cca aac tgt gct act aga 878 Gln Tyr Asn Leu Asp Ile Pro His Lys Leu Pro Asn Cys Ala Thr Arg 175 180 185 cat gta gca cca cca gtc aaa aag aac tct aaa ata gtt ctg tct gaa 926 His Val Ala Pro Pro Val Lys Lys Asn Ser Lys Ile Val Leu Ser Glu 190 195 200 gat tac aag aag ctt tat gat atc ttc gga tca ccc ttt atg gga aat 974 Asp Tyr Lys Lys Leu Tyr Asp Ile Phe Gly Ser Pro Phe Met Gly Asn 205 210 215 220 ggt gac tgt ctt agc aaa tgc ttt gac act ctt cat ttt atc gct gct 1022 Gly Asp Cys Leu Ser Lys Cys Phe Asp Thr Leu His Phe Ile Ala Ala 225 230 235 act ctt aga tgc ccg tgt ggt tct gaa agt agc ggc gtt gga gat tgg 1070 Thr Leu Arg Cys Pro Cys Gly Ser Glu Ser Ser Gly Val Gly Asp Trp 240 245 250 act ggt ttt aag act gcc tgt tgt ggt ctt tct ggc aaa gtt aag ggt 1118 Thr Gly Phe Lys Thr Ala Cys Cys Gly Leu Ser Gly Lys Val Lys Gly 255 260 265 gtc act ttg ggt gat att aag cct ggt gat gct gtt gtc act agt atg 1166 Val Thr Leu Gly Asp Ile Lys Pro Gly Asp Ala Val Val Thr Ser Met 270 275 280 agc gca ggt aag gga gtt aag ttc ttt gcc aat tgt gtt ctt caa tat 1214 Ser Ala Gly Lys Gly Val Lys Phe Phe Ala Asn Cys Val Leu Gln Tyr 285 290 295 300 gct ggt gat gtt gaa ggt gtc tcc atc tgg aaa gtt att aaa act ttt 1262 Ala Gly Asp Val Glu Gly Val Ser Ile Trp Lys Val Ile Lys Thr Phe 305 310 315 aca gtt gat gag act gta tgc acc cct ggt ttt gaa ggc gaa ttg aac 1310 Thr Val Asp Glu Thr Val Cys Thr Pro Gly Phe Glu Gly Glu Leu Asn 320 325 330 gac ttc atc aaa cct gag agc aaa tca cta gtt gca tgc agc gtt aaa 1358 Asp Phe Ile Lys Pro Glu Ser Lys Ser Leu Val Ala Cys Ser Val Lys 335 340 345 aga gca ttc att act ggt gat att gat gat gct gta cat gat tgt atc 1406 Arg Ala Phe Ile Thr Gly Asp Ile Asp Asp Ala Val His Asp Cys Ile 350 355 360 att aca gga aaa ttg gat ctt agt acc aac ctt ttt ggt aat gtt ggt 1454 Ile Thr Gly Lys Leu Asp Leu Ser Thr Asn Leu Phe Gly Asn Val Gly 365 370 375 380 cta tta ttc aag aag act cca tgg ttt gta caa aag tgt ggt gca ctt 1502 Leu Leu Phe Lys Lys Thr Pro Trp Phe Val Gln Lys Cys Gly Ala Leu 385 390 395 ttt gta gac gct tgg aaa gta gta gag gag ctt tgt ggt tca ctc aca 1550 Phe Val Asp Ala Trp Lys Val Val Glu Glu Leu Cys Gly Ser Leu Thr 400 405 410 ctt aca tac aag caa att tat gaa gtt gta gca tca ctt tgc act tct 1598 Leu Thr Tyr Lys Gln Ile Tyr Glu Val Val Ala Ser Leu Cys Thr Ser 415 420 425 gct ttt acg att gta aac tac aag cca aca ttt gtg gtt cca gac aat 1646 Ala Phe Thr Ile Val Asn Tyr Lys Pro Thr Phe Val Val Pro Asp Asn 430 435 440 cgt gtt aaa gat ctt gta gac aag tgt gtg aaa gtt ctt gta aaa gca 1694 Arg Val Lys Asp Leu Val Asp Lys Cys Val Lys Val Leu Val Lys Ala 445 450 455 460 ttt gat gtt ttt acg cag att atc aca ata gct ggt att gag gcc aaa 1742 Phe Asp Val Phe Thr Gln Ile Ile Thr Ile Ala Gly Ile Glu Ala Lys 465 470 475 tgc ttt gtg ctt ggt gct aaa tac ctg ttg ttc aat aat gca ctt gtc 1790 Cys Phe Val Leu Gly Ala Lys Tyr Leu Leu Phe Asn Asn Ala Leu Val 480 485 490 aaa ctt gtc agt gtt aaa atc ctt ggc aag aag caa aag ggt ctt gaa 1838 Lys Leu Val Ser Val Lys Ile Leu Gly Lys Lys Gln Lys Gly Leu Glu 495 500 505 tgt gca ttc ttt gct act agc ttg gtt ggt gca act gtt aat gtg aca 1886 Cys Ala Phe Phe Ala Thr Ser Leu Val Gly Ala Thr Val Asn Val Thr 510 515 520 cct aaa aga aca gag act gcc act atc agc ttg aac aag gtt gat gat 1934 Pro Lys Arg Thr Glu Thr Ala Thr Ile Ser Leu Asn Lys Val Asp Asp 525 530 535 540 gtt gta gca cca gga gag ggt tat atc gtc att gtt ggt gat atg gct 1982 Val Val Ala Pro Gly Glu Gly Tyr Ile Val Ile Val Gly Asp Met Ala 545 550 555 ttc tac aag agt ggt gaa tat tat ttc atg atg tct agt cct aat ttt 2030 Phe Tyr Lys Ser Gly Glu Tyr Tyr Phe Met Met Ser Ser Pro Asn Phe 560 565 570 gtt ctt act aac aat gtt ttt aaa gca gtt aaa gtt cca tct tat gac 2078 Val Leu Thr Asn Asn Val Phe Lys Ala Val Lys Val Pro Ser Tyr Asp 575 580 585 atc gtt tat gat gtt gat aat gat acc aaa agc aaa atg att gca aaa 2126 Ile Val Tyr Asp Val Asp Asn Asp Thr Lys Ser Lys Met Ile Ala Lys 590 595 600 ctt ggt tca tca ttt gaa caa ata cca act ggc aca caa gat cca att 2174 Leu Gly Ser Ser Phe Glu Gln Ile Pro Thr Gly Thr Gln Asp Pro Ile 605 610 615 620 cgg ttc tgt att gaa aat gaa gtt tgt gtt gtc tgt ggt tgt tgg ctt 2222 Arg Phe Cys Ile Glu Asn Glu Val Cys Val Val Cys Gly Cys Trp Leu 625 630 635 aac aat ggt tgc atg tgc gat cgt act tct atg cag agt ttt act gtt 2270 Asn Asn Gly Cys Met Cys Asp Arg Thr Ser Met Gln Ser Phe Thr Val 640 645 650 gat caa agt tat tta aac gag tgc ggg gtt cta gtg cag ctc gac 2315 Asp Gln Ser Tyr Leu Asn Glu Cys Gly Val Leu Val Gln Leu Asp 655 660 665 tagaaccctg caatggtact gatccagacc atgttagtag agcttttgac atctacaaca 2375 aagatgttgc gtgtattggt aaattcctta agacgaattg ttcaagattt aggaatttgg 2435 acaaacatga tgcctactac attgtcaaac gttgtacaaa gaccgttatg gaccatgagc 2495 aagtctgtta taacgatctt aaagattctg gtgctgttgc tgagcatgac ttcttcacat 2555 ataaagaggg tagatgtgag ttcggtaatg ttgcacgtag gaatcttaca aagtacacaa 2615 tgatggatct ttgttacgct atcagaaatt ttgatgaaaa gaactgtgaa gttctcaaag 2675 aaatactcgt gacagtaggt gcttgcactg aagaattctt tgaaaataaa gattggtttg 2735 atccagttga aaatgaagcc atacatgaag tttatgcaaa acttggaccc attgtagcca 2795 atgctatgct taaatgtgtt gctttttgcg atgcgatagt ggaaaaaggc tatataggtg 2855 ttataacact tgacaaccaa gatcttaatg gcaatttcta cgatttcggc gatttcgtga 2915 agactgctcc gggttttggt tgcgcttgtg ttacatcata ttattcttat atgatgcctt 2975 taatggggat gacttcatgc ttagagtctg aaaactttgt gaaaagtgac atctatggtt 3035 ctgattataa gcagtatgat ttactagctt atgattttac cgaacataag gagtaccttt 3095 tccaaaaata ctttaagtac tgggatcgca catatcaccc aaattgttct gattgtacta 3155 gtgacgagtg tattattcat tgtgctaatt ttaacacatt gttttctatg acaataccaa 3215 tgacagcttt tggaccactt gtccgtaaag ttcatattga tggtgtacca gtagttgtta 3275 ctgcaggtta ccatttcaaa caacttggta tagtatggaa tcttgatgta aaattagaca 3335 caatgaagtt gagcatgact gatcttctta gatttgtcac agatccaaca cttcttgtag 3395 catcaagccc tgcactttta gaccagcgta ctgtctgttt ctccattgca gctttgagta 3455 ctggtattac atatcagaca gtaaaaccag gtcactttaa caaggatttc tacgatttca 3515 taacagagcg tggattcttt gaagagggat ctgagttaac attaaaacat tttttctttg 3575 cacagggtgg tgaagctgct atgacagact tcaattatta tcgctacaat agagtcacag 3635 tacttgatat ttgccaagct caatttgttt acaaaatagt tggcaagtat tttgaatgtt 3695 atgacggtgg gtgcattaat gctcgtgaag ttgttgttac aaactatgac aagagtgctg 3755 gctatccttt gaacaaattt ggtaaagcta gactttacta cgaaactctt tcatatgaag 3815 agcaggatgc actttttgct ttaacaaaga gaaatgtttt acccacaatg actcaaatga 3875 atttgaaata cgctatttct ggtaaggcaa gagctcgtac agtaggagga gtttcacttc 3935 tttctaccat gactacgaga caatatcatc agaagcattt gaagtcaatt gctgcaacac 3995 gcaatgctac tgtggtcatt ggttcaacca agttttatgg tggttgggac aatatgctta 4055 aaaatttaat gcgtgatgtt gataatggtt gtttgatggg atgggactat cctaagtgtg 4115 accgtgcttt acctaatatg attagaatgg cttctgccat gatattaggt tctaagcatg 4175 ttggttgttg tacacataat gataggttct accgcctctc caatgagtta gctcaagtac 4235 tcacagaagt tgtgcattgc acaggtggtt tttattttaa acctggtggt acaactagcg 4295 gtgatggtac tacagcatat gctaactctg cttttaacat ctttcaagct gtttctgcta 4355 atgttaataa gcttttgggg gttgattcaa acgcttgtaa caacgttaca gtaaaatcca 4415 tacaacgtaa aatttacgat aattgttatc gtagtagcag cattgatgaa gaatttgttg 4475 ttgagtactt tagttatttg agaaaacact tttctatgat gattttatct gatgatggag 4535 ttgtgtgcta caacaaagat tatgcggatt taggttatgt agctgacatt aatgctttta 4595 aagcaacact ttattaccag aataacgtct ttatgtccac ttctaagtgt tgggtagaac 4655 cagatcttag tgttggacca catgaatttt gttcacagca tacattgcag attgttgggc 4715 ctgatggaga ctactatctt ccctatccag acccgtccag aattttgtca gctggtgtgt 4775 ttgttgatga catagttaaa acagacaatg ttattatgtt agaacgttac gtgtcattgg 4835 ctattgacgc atacccgctc acaaaacacc ctaagcctgc ttatcaaaaa gtgttttaca 4895 ctctactaga ttgggttaaa catctacaga aaaatttgaa tgcaggtgtt cttgattcgt 4955 tttcagtgac aatgttagag gaaggtcaag ataagttctg gagtgaagag ttttacgcta 5015 gcctctatga aaagtccact gtcttgcaag ctgcaggcat gtgtgtagta tgtggttcgc 5075 aaactgtact tcgttgtgga gactgtctta ggagaccact tttatgcacg aaatgtgctt 5135 acgaccatgt tatgggaaca aagcataaat tcattatgtc tatcacacca tatgtgtgta 5195 gttttaatgg ttgtaatgtc aatgatgtta caaagttgtt tttaggtggt cttagttatt 5255 attgtatgaa ccacaaacca cagttgtcat tcccactctg tgctaatggc aacgtttttg 5315 gtctatataa aagtagtgca gtcggctcag aggctgttga agatttcaac aaacttgcag 5375 tttctgactg gactaatgta gaagactaca aacttgctaa caatgtcaag gaatctctga 5435 aaattttcgc tgctgaaact gtgaaagcta aggaggagtc tgttaaatct gaatatgctt 5495 atgctgtatt aaaggaggtt atcggcccta aggaaattgt actccaatgg gaagcttcta 5555 agactaagcc tccacttaac agaaattcag ttttcacgtg ttttcagata agtaaggata 5615 ctaaaattca attaggtgaa tttgtgtttg agcaatctga gtacggtagt gattctgttt 5675 attacaagag cacgagtact tacaaattga caccaggtat gatttttgtg ttgacttctc 5735 ataatgtgag tcctcttaaa gctccaattt tagtcaacca agaaaagtac aataccatat 5795 ctaagctcta tcctgtcttt aatatagcgg aggcctataa tacactggtt ccttactacc 5855 aaatgatagg taagcaaaaa tttacaacta tccaaggtcc tcctggtagc ggtaaatctc 5915 attgtgttat aggtttgggt ttgtattacc ctcaggcgag aatagtctac actgcatgtt 5975 ctcatgcggc tgtagacgct ttatgtgaaa aagcagccaa aaacttcaat gttgatagat 6035 gttcaaggat aatacctcaa agaatcagag ttgattgtta cacaggcttt aagcctaata 6095 acaccaatgc gcagtacttg ttttgtactg ttaatgctct accagaagca agttgtgaca 6155 ttgttgtagt tgatgaggtc tctatgtgta ctaattatga tcttagtgtc ataaatagcc 6215 gactgagtta caaacatatt gtttatgttg gagacccaca gcagctacca gctcctagaa 6275 ctttgattaa taagggtgta cttcaaccgc aggattacaa tgttgtaacc aaaagaatgt 6335 gcacactagg acctgatgtc tttttgcata aatgttacag gtgcccagct gaaattgtta 6395 aracagtctc tgcacttgtt tatgaaaata aatttgtacc tgtcaaccca gaatcaaagc 6455 agtgcttcaa aatgtttgta aaaggtcagr ttcagattga gtctaactct tctataaaca 6515 acaagcaact agaggttgtc aaggcctttt tagcacataa tccaaaatgg cgtaaagctg 6575 ttttcatctc accctataat agtcaaaatt atgttgctcg gcgtcttctt ggtttgcaaa 6635 cgcaaactgt ggattccgct cagggtagtg agtatgatta cgtcatctag ctgctctgaa 6695 gatttttaat cctgctgcaa ttcacgatgt gggtaatcca aaaggcatcc gttgtgctac 6755 aacaccaata ccatggtttt gttatgatcg tgatcctatt aataacaatg ttagatgtct 6815 ggattatgac tatatggtac atggtcaaat gaatggtctt atgttatttt ggaactgtaa 6875 tgtagacatg tacccagagt tttcaattgt ttgtagattt gatactcgca ctcgctctaa 6935 attgtcttta gaaggttgta atggtggtgc attgtatgtt aataaccatg ctttccacac 6995 accagcttat gatagaagag cttttgctaa gcttaaacct atgccattct tttactatga 7055 tgatagtaat tgtgaacttg ttgatgggca acctaattat gtaccactta agtcaaatgt 7115 ttgcataaca aaatgcaaca ttggtggtgc tgtctgcaag aagcatgctg ctctttacag 7175 agcgtatgtt gaggattaca acatttttat gcaggctggt tttacaatat ggtgtcctca 7235 aaactttgac acctatatgc tttggcatgg ttttgttaat agcaaagcac ttcagagtct 7295 agaaaatgtg gcttttaatg tcgttaagaa aggtgccttc accggtttaa aaggtgactt 7355 accaactgct gttattgctg acaaaataat ggtaagagat ggacctactg acaaatgtat 7415 ttttacaaat aagactagtt tacctacaaa tgtagctttt gagttatatg caaaacgcaa 7475 acttggactc acacctccat taacaatact taggaattta ggtgttgtcg caacatataa 7535 gtttgtgttg tgggattatg aagctgaacg tcctttctca aatttcacta agcaagtgtg 7595 ttcctacact gatcttgata gtgaagttgt aacatgtttt gataatagta ttgctggctc 7655 ttttgagcgt tttactacta caagagatgc agtgcttatt tctaataacg ctgtgaaagg 7715 gcttagtgcc attaaattac aatatggcct tttgaatgat ctacctgtaa gtactgttgg 7775 aaataaacct gtcacatggt atatctatgt gcgcaagaat ggtgagtacg tcgaacaaat 7835 cgatagttac tatacacagg gacgtacttt tgaaaccttc aaacctcgta gtacaatgga 7895 agaagatttt cttagtatgg atactacact cttcatccaa aagtatggtc ttgaggatta 7955 tggttttgaa cacgttgtat ttggagatgt ctctaaaact accattggtg gtatgcatct 8015 tcttatatcg caagtgcgcc ttgcaaaaat gggtttgttt tccgttcaag aatttatgaa 8075 taattctgac agtacactga aaagttgttg tattacatat gctgatgatc catcttctaa 8135 gaatgtgtgc acttatatgg acatactctt ggacgatttt gtgactatca ttaagagctt 8195 agatcttaat gttgtgtcca aagttgtgga tgtcattgta gattgtaagg catggagatg 8255 gatgttgtgg tgtgagaatt cacatattaa aaccttctat ccacaactcc aatctgctga 8315 atggaatccc ggctatagca tgcctacact gtacaaaatc cagcgtatgt gtctcgaacg 8375 gtgtaatctc tacaattatg gtgcacaagt gaaattacct gatggcatta ctactaatgt 8435 cgttaagtat actcagttgt gtcaatacct taacactact acattgtgtg taccacacaa 8495 aatgcgtgta ttgcatttag gagctgctgg tgcatctggt gttgctcctg gtagtactgt 8555 attaagaaga tggttaccag atgatgccat attggttgat aatgatttga gagattacgt 8615 ttccgacgca gacttcagtg ttacaggtga ttgtactagt ctttacatcg aagacaagtt 8675 tgatttgctc gtctctgatt tatatgatgg ctccacaaaa tcaattgacg gtgaaaacac 8735 gtcgaaagat ggtttcttta cttatattaa tggtttcatt aaagagaaac tgtcacttgg 8795 tggatctgtt gccattaaaa tcacggaatt tagttggaat aaagatttat atgaattgat 8855 tcaaagattt gagtattgga ctgtgttttg tacaagtgtt aacacgtcat catcagaagg 8915 ctttctgatt ggtattaact acttaggacc atactgtgac aaagcaatag tagatggaaa 8975 tataatgcat gccaattata tattttggag aaactctaca attatggctc tatcacataa 9035 ctcagtccta gacactccta aattcaagtg tcgttgtaac aacgcactta ttgttaattt 9095 aaaagaaaaa gaattgaatg aaatggtcat tggattacta aggaagggta agttgctcat 9155 tagaaataat ggtaagttac taaactttgg taaccacttc gttaacacac catgaaaaaa 9215 tgctgtattt attacagttt taatcttact actaattggt agactccaat tattagaaag 9275 actattactt aatcactctt tcaatcttaa aactgtcaat gactttaata tcttatatag 9335 gagtttagca gaaaccagat tactaaaagt ggtgcttcga gtaatctttc tagtcttact 9395 aggattttgc tgctacagat tgttagtcac attaatgtaa ggcaacccga tgtctaaaac 9455 tggtttttcc gaggaattac tggtcatcgc gctgtctact cttgtacaga atggtaagca 9515 cgtgtaatag gaggtacaag caaccctatt gcatattagg aagtttagat ttgatttggc 9575 aatgctagat ttagtaattt agagaagttt aaagatccgc tacgacgagc caacaatgga 9635 agagctaacg tctggatcta gtgattgttt aaaatgtaaa attgtttgaa aattttcctt 9695 ttgatagtga tacaaaaaa 9714 25 667 PRT Transmissible gastroenteritis coronavirus MOD_RES (108) Gly or Val 25 Met Ser Ser Lys Gln Phe Lys Ile Leu Val Asn Glu Asp Tyr Gln Val 1 5 10 15 Asn Val Pro Ser Leu Pro Ile Arg Asp Val Leu Gln Glu Ile Lys Tyr 20 25 30 Cys Tyr Arg Asn Gly Phe Glu Gly Tyr Val Phe Val Pro Glu Tyr Cys 35 40 45 Arg Asp Leu Val Asp Cys Asp Arg Lys Asp His Tyr Val Ile Gly Val 50 55 60 Leu Gly Asn Gly Val Ser Asp Leu Lys Pro Val Leu Leu Thr Glu Pro 65 70 75 80 Ser Val Met Leu Gln Gly Phe Ile Val Arg Ala Asn Cys Asn Gly Val 85 90 95 Leu Glu Asp Phe Asp Leu Lys Ile Ala Arg Thr Xaa Arg Gly Ala Ile 100 105 110 Tyr Val Asp Gln Tyr Met Cys Gly Ala Asp Gly Lys Pro Val Ile Glu 115 120 125 Gly Asp Phe Lys Asp Tyr Phe Gly Asp Glu Asp Ile Ile Glu Phe Glu 130 135 140 Gly Glu Glu Tyr His Cys Ala Trp Thr Thr Val Arg Asp Glu Lys Pro 145 150 155 160 Leu Asn Gln Gln Thr Leu Phe Thr Ile Gln Glu Ile Gln Tyr Asn Leu 165 170 175 Asp Ile Pro His Lys Leu Pro Asn Cys Ala Thr Arg His Val Ala Pro 180 185 190 Pro Val Lys Lys Asn Ser Lys Ile Val Leu Ser Glu Asp Tyr Lys Lys 195 200 205 Leu Tyr Asp Ile Phe Gly Ser Pro Phe Met Gly Asn Gly Asp Cys Leu 210 215 220 Ser Lys Cys Phe Asp Thr Leu His Phe Ile Ala Ala Thr Leu Arg Cys 225 230 235 240 Pro Cys Gly Ser Glu Ser Ser Gly Val Gly Asp Trp Thr Gly Phe Lys 245 250 255 Thr Ala Cys Cys Gly Leu Ser Gly Lys Val Lys Gly Val Thr Leu Gly 260 265 270 Asp Ile Lys Pro Gly Asp Ala Val Val Thr Ser Met Ser Ala Gly Lys 275 280 285 Gly Val Lys Phe Phe Ala Asn Cys Val Leu Gln Tyr Ala Gly Asp Val 290 295 300 Glu Gly Val Ser Ile Trp Lys Val Ile Lys Thr Phe Thr Val Asp Glu 305 310 315 320 Thr Val Cys Thr Pro Gly Phe Glu Gly Glu Leu Asn Asp Phe Ile Lys 325 330 335 Pro Glu Ser Lys Ser Leu Val Ala Cys Ser Val Lys Arg Ala Phe Ile 340 345 350 Thr Gly Asp Ile Asp Asp Ala Val His Asp Cys Ile Ile Thr Gly Lys 355 360 365 Leu Asp Leu Ser Thr Asn Leu Phe Gly Asn Val Gly Leu Leu Phe Lys 370 375 380 Lys Thr Pro Trp Phe Val Gln Lys Cys Gly Ala Leu Phe Val Asp Ala 385 390 395 400 Trp Lys Val Val Glu Glu Leu Cys Gly Ser Leu Thr Leu Thr Tyr Lys 405 410 415 Gln Ile Tyr Glu Val Val Ala Ser Leu Cys Thr Ser Ala Phe Thr Ile 420 425 430 Val Asn Tyr Lys Pro Thr Phe Val Val Pro Asp Asn Arg Val Lys Asp 435 440 445 Leu Val Asp Lys Cys Val Lys Val Leu Val Lys Ala Phe Asp Val Phe 450 455 460 Thr Gln Ile Ile Thr Ile Ala Gly Ile Glu Ala Lys Cys Phe Val Leu 465 470 475 480 Gly Ala Lys Tyr Leu Leu Phe Asn Asn Ala Leu Val Lys Leu Val Ser 485 490 495 Val Lys Ile Leu Gly Lys Lys Gln Lys Gly Leu Glu Cys Ala Phe Phe 500 505 510 Ala Thr Ser Leu Val Gly Ala Thr Val Asn Val Thr Pro Lys Arg Thr 515 520 525 Glu Thr Ala Thr Ile Ser Leu Asn Lys Val Asp Asp Val Val Ala Pro 530 535 540 Gly Glu Gly Tyr Ile Val Ile Val Gly Asp Met Ala Phe Tyr Lys Ser 545 550 555 560 Gly Glu Tyr Tyr Phe Met Met Ser Ser Pro Asn Phe Val Leu Thr Asn 565 570 575 Asn Val Phe Lys Ala Val Lys Val Pro Ser Tyr Asp Ile Val Tyr Asp 580 585 590 Val Asp Asn Asp Thr Lys Ser Lys Met Ile Ala Lys Leu Gly Ser Ser 595 600 605 Phe Glu Gln Ile Pro Thr Gly Thr Gln Asp Pro Ile Arg Phe Cys Ile 610 615 620 Glu Asn Glu Val Cys Val Val Cys Gly Cys Trp Leu Asn Asn Gly Cys 625 630 635 640 Met Cys Asp Arg Thr Ser Met Gln Ser Phe Thr Val Asp Gln Ser Tyr 645 650 655 Leu Asn Glu Cys Gly Val Leu Val Gln Leu Asp 660 665 26 1470 PRT Transmissible gastroenteritis coronavirus MOD_RES (1405) Ile or Val 26 Ser Lys Leu Phe Lys Arg Val Arg Gly Ser Ser Ala Ala Arg Leu Glu 1 5 10 15 Pro Cys Asn Gly Thr Asp Pro Asp His Val Ser Arg Ala Phe Asp Ile 20 25 30 Tyr Asn Lys Asp Val Ala Cys Ile Gly Lys Phe Leu Lys Thr Asn Cys 35 40 45 Ser Arg Phe Arg Asn Leu Asp Lys His Asp Ala Tyr Tyr Ile Val Lys 50 55 60 Arg Cys Thr Lys Thr Val Met Asp His Glu Gln Val Cys Tyr Asn Asp 65 70 75 80 Leu Lys Asp Ser Gly Ala Val Ala Glu His Asp Phe Phe Thr Tyr Lys 85 90 95 Glu Gly Arg Cys Glu Phe Gly Asn Val Ala Arg Arg Asn Leu Thr Lys 100 105 110 Tyr Thr Met Met Asp Leu Cys Tyr Ala Ile Arg Asn Phe Asp Glu Lys 115 120 125 Asn Cys Glu Val Leu Lys Glu Ile Leu Val Thr Val Gly Ala Cys Thr 130 135 140 Glu Glu Phe Phe Glu Asn Lys Asp Trp Phe Asp Pro Val Glu Asn Glu 145 150 155 160 Ala Ile His Glu Val Tyr Ala Lys Leu Gly Pro Ile Val Ala Asn Ala 165 170 175 Met Leu Lys Cys Val Ala Phe Cys Asp Ala Ile Val Glu Lys Gly Tyr 180 185 190 Ile Gly Val Ile Thr Leu Asp Asn Gln Asp Leu Asn Gly Asn Phe Tyr 195 200 205 Asp Phe Gly Asp Phe Val Lys Thr Ala Pro Gly Phe Gly Cys Ala Cys 210 215 220 Val Thr Ser Tyr Tyr Ser Tyr Met Met Pro Leu Met Gly Met Thr Ser 225 230 235 240 Cys Leu Glu Ser Glu Asn Phe Val Lys Ser Asp Ile Tyr Gly Ser Asp 245 250 255 Tyr Lys Gln Tyr Asp Leu Leu Ala Tyr Asp Phe Thr Glu His Lys Glu 260 265 270 Tyr Leu Phe Gln Lys Tyr Phe Lys Tyr Trp Asp Arg Thr Tyr His Pro 275 280 285 Asn Cys Ser Asp Cys Thr Ser Asp Glu Cys Ile Ile His Cys Ala Asn 290 295 300 Phe Asn Thr Leu Phe Ser Met Thr Ile Pro Met Thr Ala Phe Gly Pro 305 310 315 320 Leu Val Arg Lys Val His Ile Asp Gly Val Pro Val Val Val Thr Ala 325 330 335 Gly Tyr His Phe Lys Gln Leu Gly Ile Val Trp Asn Leu Asp Val Lys 340 345 350 Leu Asp Thr Met Lys Leu Ser Met Thr Asp Leu Leu Arg Phe Val Thr 355 360 365 Asp Pro Thr Leu Leu Val Ala Ser Ser Pro Ala Leu Leu Asp Gln Arg 370 375 380 Thr Val Cys Phe Ser Ile Ala Ala Leu Ser Thr Gly Ile Thr Tyr Gln 385 390 395 400 Thr Val Lys Pro Gly His Phe Asn Lys Asp Phe Tyr Asp Phe Ile Thr 405 410 415 Glu Arg Gly Phe Phe Glu Glu Gly Ser Glu Leu Thr Leu Lys His Phe 420 425 430 Phe Phe Ala Gln Gly Gly Glu Ala Ala Met Thr Asp Phe Asn Tyr Tyr 435 440 445 Arg Tyr Asn Arg Val Thr Val Leu Asp Ile Cys Gln Ala Gln Phe Val 450 455 460 Tyr Lys Ile Val Gly Lys Tyr Phe Glu Cys Tyr Asp Gly Gly Cys Ile 465 470 475 480 Asn Ala Arg Glu Val Val Val Thr Asn Tyr Asp Lys Ser Ala Gly Tyr 485 490 495 Pro Leu Asn Lys Phe Gly Lys Ala Arg Leu Tyr Tyr Glu Thr Leu Ser 500 505 510 Tyr Glu Glu Gln Asp Ala Leu Phe Ala Leu Thr Lys Arg Asn Val Leu 515 520 525 Pro Thr Met Thr Gln Met Asn Leu Lys Tyr Ala Ile Ser Gly Lys Ala 530 535 540 Arg Ala Arg Thr Val Gly Gly Val Ser Leu Leu Ser Thr Met Thr Thr 545 550 555 560 Arg Gln Tyr His Gln Lys His Leu Lys Ser Ile Ala Ala Thr Arg Asn 565 570 575 Ala Thr Val Val Ile Gly Ser Thr Lys Phe Tyr Gly Gly Trp Asp Asn 580 585 590 Met Leu Lys Asn Leu Met Arg Asp Val Asp Asn Gly Cys Leu Met Gly 595 600 605 Trp Asp Tyr Pro Lys Cys Asp Arg Ala Leu Pro Asn Met Ile Arg Met 610 615 620 Ala Ser Ala Met Ile Leu Gly Ser Lys His Val Gly Cys Cys Thr His 625 630 635 640 Asn Asp Arg Phe Tyr Arg Leu Ser Asn Glu Leu Ala Gln Val Leu Thr 645 650 655 Glu Val Val His Cys Thr Gly Gly Phe Tyr Phe Lys Pro Gly Gly Thr 660 665 670 Thr Ser Gly Asp Gly Thr Thr Ala Tyr Ala Asn Ser Ala Phe Asn Ile 675 680 685 Phe Gln Ala Val Ser Ala Asn Val Asn Lys Leu Leu Gly Val Asp Ser 690 695 700 Asn Ala Cys Asn Asn Val Thr Val Lys Ser Ile Gln Arg Lys Ile Tyr 705 710 715 720 Asp Asn Cys Tyr Arg Ser Ser Ser Ile Asp Glu Glu Phe Val Val Glu 725 730 735 Tyr Phe Ser Tyr Leu Arg Lys His Phe Ser Met Met Ile Leu Ser Asp 740 745 750 Asp Gly Val Val Cys Tyr Asn Lys Asp Tyr Ala Asp Leu Gly Tyr Val 755 760 765 Ala Asp Ile Asn Ala Phe Lys Ala Thr Leu Tyr Tyr Gln Asn Asn Val 770 775 780 Phe Met Ser Thr Ser Lys Cys Trp Val Glu Pro Asp Leu Ser Val Gly 785 790 795 800 Pro His Glu Phe Cys Ser Gln His Thr Leu Gln Ile Val Gly Pro Asp 805 810 815 Gly Asp Tyr Tyr Leu Pro Tyr Pro Asp Pro Ser Arg Ile Leu Ser Ala 820 825 830 Gly Val Phe Val Asp Asp Ile Val Lys Thr Asp Asn Val Ile Met Leu 835 840 845 Glu Arg Tyr Val Ser Leu Ala Ile Asp Ala Tyr Pro Leu Thr Lys His 850 855 860 Pro Lys Pro Ala Tyr Gln Lys Val Phe Tyr Thr Leu Leu Asp Trp Val 865 870 875 880 Lys His Leu Gln Lys Asn Leu Asn Ala Gly Val Leu Asp Ser Phe Ser 885 890 895 Val Thr Met Leu Glu Glu Gly Gln Asp Lys Phe Trp Ser Glu Glu Phe 900 905 910 Tyr Ala Ser Leu Tyr Glu Lys Ser Thr Val Leu Gln Ala Ala Gly Met 915 920 925 Cys Val Val Cys Gly Ser Gln Thr Val Leu Arg Cys Gly Asp Cys Leu 930 935 940 Arg Arg Pro Leu Leu Cys Thr Lys Cys Ala Tyr Asp His Val Met Gly 945 950 955 960 Thr Lys His Lys Phe Ile Met Ser Ile Thr Pro Tyr Val Cys Ser Phe 965 970 975 Asn Gly Cys Asn Val Asn Asp Val Thr Lys Leu Phe Leu Gly Gly Leu 980 985 990 Ser Tyr Tyr Cys Met Asn His Lys Pro Gln Leu Ser Phe Pro Leu Cys 995 1000 1005 Ala Asn Gly Asn Val Phe Gly Leu Tyr Lys Ser Ser Ala Val Gly Ser 1010 1015 1020 Glu Ala Val Glu Asp Phe Asn Lys Leu Ala Val Ser Asp Trp Thr Asn 1025 1030 1035 1040 Val Glu Asp Tyr Lys Leu Ala Asn Asn Val Lys Glu Ser Leu Lys Ile 1045 1050 1055 Phe Ala Ala Glu Thr Val Lys Ala Lys Glu Glu Ser Val Lys Ser Glu 1060 1065 1070 Tyr Ala Tyr Ala Val Leu Lys Glu Val Ile Gly Pro Lys Glu Ile Val 1075 1080 1085 Leu Gln Trp Glu Ala Ser Lys Thr Lys Pro Pro Leu Asn Arg Asn Ser 1090 1095 1100 Val Phe Thr Cys Phe Gln Ile Ser Lys Asp Thr Lys Ile Gln Leu Gly 1105 1110 1115 1120 Glu Phe Val Phe Glu Gln Ser Glu Tyr Gly Ser Asp Ser Val Tyr Tyr 1125 1130 1135 Lys Ser Thr Ser Thr Tyr Lys Leu Thr Pro Gly Met Ile Phe Val Leu 1140 1145 1150 Thr Ser His Asn Val Ser Pro Leu Lys Ala Pro Ile Leu Val Asn Gln 1155 1160 1165 Glu Lys Tyr Asn Thr Ile Ser Lys Leu Tyr Pro Val Phe Asn Ile Ala 1170 1175 1180 Glu Ala Tyr Asn Thr Leu Val Pro Tyr Tyr Gln Met Ile Gly Lys Gln 1185 1190 1195 1200 Lys Phe Thr Thr Ile Gln Gly Pro Pro Gly Ser Gly Lys Ser His Cys 1205 1210 1215 Val Ile Gly Leu Gly Leu Tyr Tyr Pro Gln Ala Arg Ile Val Tyr Thr 1220 1225 1230 Ala Cys Ser His Ala Ala Val Asp Ala Leu Cys Glu Lys Ala Ala Lys 1235 1240 1245 Asn Phe Asn Val Asp Arg Cys Ser Arg Ile Ile Pro Gln Arg Ile Arg 1250 1255 1260 Val Asp Cys Tyr Thr Gly Phe Lys Pro Asn Asn Thr Asn Ala Gln Tyr 1265 1270 1275 1280 Leu Phe Cys Thr Val Asn Ala Leu Pro Glu Ala Ser Cys Asp Ile Val 1285 1290 1295 Val Val Asp Glu Val Ser Met Cys Thr Asn Tyr Asp Leu Ser Val Ile 1300 1305 1310 Asn Ser Arg Leu Ser Tyr Lys His Ile Val Tyr Val Gly Asp Pro Gln 1315 1320 1325 Gln Leu Pro Ala Pro Arg Thr Leu Ile Asn Lys Gly Val Leu Gln Pro 1330 1335 1340 Gln Asp Tyr Asn Val Val Thr Lys Arg Met Cys Thr Leu Gly Pro Asp 1345 1350 1355 1360 Val Phe Leu His Lys Cys Tyr Arg Cys Pro Ala Glu Ile Val Lys Thr 1365 1370 1375 Val Ser Ala Leu Val Tyr Glu Asn Lys Phe Val Pro Val Asn Pro Glu 1380 1385 1390 Ser Lys Gln Cys Phe Lys Met Phe Val Lys Gly Gln Xaa Gln Ile Glu 1395 1400 1405 Ser Asn Ser Ser Ile Asn Asn Lys Gln Leu Glu Val Val Lys Ala Phe 1410 1415 1420 Leu Ala His Asn Pro Lys Trp Arg Lys Ala Val Phe Ile Ser Pro Tyr 1425 1430 1435 1440 Asn Ser Gln Asn Tyr Val Ala Arg Arg Leu Leu Gly Leu Gln Thr Gln 1445 1450 1455 Thr Val Asp Ser Ala Gln Gly Ser Glu Tyr Asp Tyr Val Ile 1460 1465 1470 27 16 PRT Transmissible gastroenteritis coronavirus 27 Met Lys Lys Cys Cys Ile Tyr Tyr Ser Phe Asn Leu Thr Thr Asn Trp 1 5 10 15 28 95 RNA Transmissible gastroenteritis coronavirus 28 uuuaaacgag ugcgggguuc uagugcagcu cgacuagaac ccugcaaugg uacugaucca 60 gaccauguua guagagcuuu ugacaucuac aacaa 95 29 207 RNA Transmissible gastroenteritis coronavirus modified_base (57)..(198) a, u, c, g, other or unknown 29 uuuaaacgag ugcgggguuc uagugcagcu cgacuagaac ccugcaaugg uacugannnn 60 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180 nnnnnnnnnn nnnnnnnngu uauggac 207 30 19 RNA Transmissible gastroenteritis coronavirus 30 caugagcaag ucuguuaua 19 31 20 DNA Transmissible gastroenteritis coronavirus 31 taatacgact cactataggg 20 32 19 DNA Transmissible gastroenteritis coronavirus 32 aaaaaaaggg tcggcatgg 19 

1. A defective viral genome comprising parental virus genome having viral replicase recognition signals located on ends 3′ and 5′, further comprising internal deletions, and Wherein said defective viral genome depends on a helper virus for its replication.
 2. A genome according to claim 1, comprising, additionally, the complete sequence coding for the parental virus replicase.
 3. A genome according to the preceding claims, wherein the aforesaid parental virus is a coronavirus.
 4. A genome according to the preceding claims, comprising a defective genome of a porcine, canine or feline coronavirus.
 5. A genome according to claim 4, comprising a defective genome of the virus of transmissible gastroenteritis of pigs (TGEV).
 6. A genome according to any of the preceding claims, wherein the aforesaid helper virus is the parental virus.
 7. An expression vector based on a recombinant defective viral genome expressing at least one antigen capable of inducing systemic and secretory immune responses, comprising a defective viral genome according to claims 1 to 6, or its corresponding complementary DNA (cDNA), and, at least, one DNA sequence coding for an antigen capable of conferring systemic and mucosal immunity.
 8. A vector according to claim 7, comprising more than one DNA sequence, each one of which codes for a different antigen capable of conferring systemic and mucosal immunity.
 9. An expression vector based on a recombinant defective viral genome expressing at least one antibody which confers protection against an infectious agent, comprising a defective viral genome according to claims 1 to 6, or its corresponding complementary DNA (cDNA), and, at least, one DNA sequence coding for an antibody that confers protection against one infectious agent.
 10. A vector according to claim 9, comprising more than one DNA sequence, each one of which codes for a different antibody that confers protection against an infectious agent.
 11. A recombinant system based on recombinant defective viruses expressing, at least, one antigen capable of inducing systemic and mucosal immunity, comprising: a) a recombinant expression vector based on a defective viral genome according to claims 7 or 8, containing, at least, one DNA sequence coding for an antigen capable of conferring systemic and mucosal immunity; and b) a helper virus.
 12. A system according to claim 11, wherein the aforesaid expression vector comprises more than one DNA sequence, each one of which codes for a different antigen capable of conferring systemic and mucosal immunity.
 13. A system according to claim 11, comprising different expression vectors each one of which contains a different DNA sequence coding for a different antigen capable of conferring systemic and mucosal immunity.
 14. A system according to any of claims 11 to 13, wherein the aforesaid helper virus is the parental virus from which the defective viral genome is derived.
 15. A system according to claim 14, wherein the aforesaid helper virus provides the functional and structural proteins for the replication and encapsidation of the defective genome.
 16. A system according to claim 14, wherein the aforesaid helper virus provides the structural proteins for the encapsidation of the defective genome.
 17. A recombinant system based on recombinant defective viruses expressing, at least, one antibody that confers protection against an infectious agent, comprising: a) a recombinant expression vector based on a defective viral genome according to claims 9 or 10, containing, at least, one DNA, sequence coding for an antibody that confers protection against an infectious agent, and b) a helper virus.
 18. A system according to claim 17, wherein, the aforesaid expression vector comprises more than one DNA sequence, each one of which codes for a different antibody conferring protection against and infectious agent.
 19. A system according to claim 17, comprising different expression vectors each one of which contains a different DNA sequence coding for an antibody that confers protection against an infectious agent.
 20. A system according to any of claims 17 to 19, wherein the aforesaid helper virus is the parental virus from which the defective viral genome is derived.
 21. A system according to claim 20, wherein the aforesaid helper virus provides the functional and structural proteins for the replication and encapsidation of the defective genome.
 22. A system according to claim 20, wherein the aforesaid helper virus provides the structural proteins for the encapsidation of the defective genome.
 23. A vaccine capable of inducing protection in animals against an infectious agent, comprising a suitable quantity of a recombinant system according to any of claims 11 to 22, together with a pharmaceutically acceptable excipient.
 24. A vaccine according to claim 23, wherein the aforesaid recombinant system comprises an expression vector containing a DNA sequence coding for an antigen capable conferring systemic and mucosal immunity.
 25. A vaccine according to claim 23, wherein the aforesaid recombinant system comprises an expression vector containing more than one DNA sequence, each one of which codes for different antigen capable of conferring systemic and mucosal immunity.
 26. A vaccine according to claim 23, wherein the aforesaid recombinant system comprises different expression vectors each one of which contains at least one different DNA sequence coding for a different antigen capable of conferring systemic and mucosal immunity.
 27. A vaccine according to claim 23, suitable for the conferring of immunity against porcine infectious agents, wherein the recombinant system comprises at least one expression vector containing at least one DNA sequence coding for a porcine pathogen antigen.
 28. A vaccine according to claim 23, suitable for the conferring of immunity against porcine infectious agents, wherein the recombinant system comprises one expression vector containing more than one DNA sequence, each one of which codes for a different porcine pathogen antigen.
 29. A vaccine according to claim 23, suitable for the conferring of immunity against porcine infectious agents, wherein the recombinant system comprises different expression vectors each one of which contains at least one DNA sequence coding for a porcine pathogen antigen.
 30. A vaccine according to claims 27 to 29, wherein the aforesaid porcine pathogen is selected from a group consisting essentially of: Actinobacillus suis, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Porcine parvovirus, Leptospira, Escherichia coli, Erysipelothrix rhusiopathiae, Pasteurella multocida, Bordetella bronchiseptica, Clostridium sp., Serpulina hyodysenteriae, Mycoplasma hyopneumoniae, porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus, rotavirus, or against the pathogens causative of porcine reproductive and respiratory syndrome, Aujeszky's disease (pseudorabies), swine influenza or transmissible gastroenteritis and the etiologic agents of atrophic rhinitis and proliferative ileitis.
 31. A vaccine according to claim 23, suitable for the conferring of immunity against canine infectious agents, wherein the recombinant system comprises at least one expression vector containing at least one DNA sequence coding for a canine pathogen antigen.
 32. A vaccine according to claim 23, suitable for the conferring of immunity against canine infectious agents, wherein the recombinant system comprises an expression vector containing more than one DNA sequence, each one of which codes for a different canine pathogen antigen.
 33. A vaccine according to claim, 23, suitable for the conferring of immunity against canine infectious agents, wherein the recombinant system comprises different expression vectors each one of which contains at least one DNA sequence coding for a canine pathogen antigen.
 34. A vaccine according to claims 31 to 33, wherein the aforesaid canine pathogen is selected from a group constituted essentially of: canine herpesviruses, canine adenovirus types 1 and 2, canine parvovirus types 1 and 2, canine reovirus, canine rotavirus, canine coronavirus, canine parainfluenza virus, canine influenza virus, distemper virus, rabies virus, retovirus and canine calicivirus.
 35. A vaccine according to claim 23, suitable for the conferring of immunity against feline infectious agents, herein the recombinant system comprises at least one expression vector containing at least one DNA sequence coding for a feline pathogen antigen.
 36. A vaccine according to claim 23, suitable for the conferring of immunity against feline infectious agents, wherein the recombinant system comprises one expression vector containing more than one DNA sequence, each one of which codes for a feline pathogen antigen.
 37. A vaccine according to claim 23, suitable for the conferring of immunity against feline infectious agents, wherein the recombinant system comprises different expression vectors each one of which contains at least one DNA sequence coding for one feline pathogen antigen.
 38. A vaccine according to claims 35 to 37 wherein the aforesaid feline pathogen is selected from a group constituted essentially of: feline calicivirus, feline immunodeficiency virus, feline herpesviruses, feline panleukopenia virus, feline reovirus, feline rotavirus, feline coronavirus, feline infectious peritonitis virus, rabies virus, feline Chlamydia psittaci and feline leukemia virus.
 39. A vaccine according to claim 23, wherein the aforesaid recombinant system comprises an expression vector containing a DNA sequence coding for an antibody that confers protection against an infectious agent.
 40. A vaccine according to claim 23, wherein the said recombinant system comprises an expression vector containing more than one DNA sequence, each one of which codes for an antibody conferring protection against an infectious agent.
 41. A vaccine according to claim 23, wherein the said recombinant system comprises different expression vectors each one of which contains at least a different DNA sequence coding for an antigen that confers protection against an infectious agent.
 42. A vaccine according to claim 23, suitable for the conferring of immunity against a porcine infectious agent, wherein the recombinant system comprises at least one expression vector containing at least one DNA sequence coding for an antibody that confers protection against the aforesaid porcine infectious agent.
 43. A vaccine according to claim 42, wherein the recombinant system comprises at least one expression vector containing at least one DNA sequence coding for the monoclonal antibody identified as 6A.C3 capable of neutralizing the virus of transmissible gastroenteritis of pigs (TGEV).
 44. A vaccine according claim 23, wherein the helper virus of the recombinant system comprises a coronavirus.
 45. A vaccine according to claim 44 wherein the aforesaid coronavirus is selected from the group composed of porcine, canine and feline coronaviruses. 